Mutant xylanase, manufacturing method and use therefor, and method for manufacturing saccharified lignocellulose

ABSTRACT

What is aimed at is provision of an inexpensive and efficient saccharification method for lignocellulose using a thermostable xylanase and provision of a mutant xylanase that has a substitute amino acid residue, and that exhibits stable activity even under severe conditions in which enzymes easily inactivate, and that provides an initial rate of reaction not significantly reduced as compared to a wild-type xylanase corresponding to the mutant xylanase. Provided is a method of producing a saccharified product of lignocellulose, including contacting a lignocellulosic raw material with a thermostable xylanase, and a mutant xylanase that provides an initial rate of reaction that is at least 70% of that provided by a wild-type xylanase corresponding thereto, that has a xylanase activity after heat treatment at 50° C. for 24 hours that is at least 50% of its xylanase activity before the heat treatment, and that has a substitute amino acid residue.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/360,514, filed May 23, 2014, which is the National Phase ofPCT/JP2012/080387, filed Nov. 22, 2012, which claims priority toJapanese Application No. 2011-257389, filed Nov. 25, 2011 and JapaneseApplication No. 2012-099096, filed Apr. 24, 2012 the disclosures ofwhich are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a method of efficiently producing asaccharified product from a lignocellulosic raw material. The inventionalso relates to a mutant xylanase, a method of producing the mutantxylanase, and uses of the mutant xylanase.

BACKGROUND ART

Xylanase is an enzyme that randomly hydrolyzes β-1,4 bonds of xylan,which is a component of plant cell walls. The enzyme is expected to beused in a wide range of applications, such as a) saccharification oflignocellulosic raw materials, b) pulp bleaching, c) animal feedadditives, d) detergent aids, and e) bread-making modifiers.

With regard to a) saccharification of lignocellulosic raw materials, amethod for saccharification of a lignocellulosic raw material is knownin which a monosaccharide serving as a fermentation substrate isproduced from a lignocellulosic raw material using an enzyme. However,the expensiveness of enzymes such as cellulases and hemicellulases(xylanase or the like) that can be used for this saccharification methodhinders practical use of this saccharification method. Addressing thisproblem, reutilization of enzymes used in the saccharification methodhas been proposed as a means effective for the reduction of the cost ofthe saccharification method (see, for example, Japanese PatentApplication Laid-Open (JP-A) No. 2006-87319 (Patent Document 1),International Publication WO2011/065449 pamphlet (Patent Document 10)and WO2011/125056 pamphlet (Patent Document 11)).

Xylanase is an enzyme that breaks down hemicellulose (of which the maincomponent is β-1,4-xylan), which is one of the main components of alignocellulosic raw material. Therefore, xylanase is one of importantenzymes in a method for saccharifying a lignocellulosic raw material.However, xylanase is known to have low stability.

Meanwhile, saccharification of a lignocellulosic raw material requirestreatment in an acidic region of from pH 4.0 to pH 6.0 at a hightemperature of from 40° C. to 60° C. for a few days. Thus, the lowstability of xylanase is a hindrance to the reutilization of thisenzyme.

A heat-resistant xylanase mutant derived from Trichoderma reesei(hereinafter abbreviated to “T. reesei”) (see, for example, WO2007/115391 pamphlet (Patent Document 2) and WO 2007/115407 pamphlet(Patent Document 3)) exhibited a residual activity of 80% or higher evenafter heat treatment at from 50° C. to 70° C. for 30 minutes.

A Bacillus-derived heat-resistant xylanase (see, for example, JP-A No.2004-121257 (Patent Document 4)) is also known to exhibit a residualactivity of 90% or higher after heat treatment at 70° C. for 30 minutes.

In regard to b) pulp bleaching, it is known that the amount of bleachingagent to be used can be decreased by using xylanase in a pulp bleachingprocess.

In general, pulp bleaching in paper-making industry consists of a firststage which is a delignification treatment process (from pH 10 to 12,80° C.) of removing lignin from pulp using an enzyme, and a second stagewhich is a bleaching process. The reason for performing the bleachingprocess in the second stage as described above is that about a fewpercent of lignin remains as a coloring component in the pulp even afterthe delignification treatment using an enzyme. Addition of a process ofallowing xylanase to work, in addition to the delignification treatmentprocess and the bleaching process, enables the breakage of hemicellulosechains bound to lignin and cellulose. As a result of this, lignin can beeffectively removed, and it is expected that an effect in terms ofdecreasing the amount of bleaching agent to be used in the bleachingprocess can be obtained.

In order to efficiently perform the process of allowing xylanase towork, it is necessary to use a xylanase having properties such that thexylanase can tolerate treatment at about pH 10 and from 70° C. to 80° C.carried out for a few hours.

A heat-resistant xylanase mutant derived from T. reesei (see, forexample, Patent Documents 2 and 3, and WO 2001/92487 (Patent Document 5)and WO 2003/046169 (Patent Document 6)) has an optimum reactiontemperature of about 70° C. and an optimum reaction pH of from 7 to 8,demonstrating the possibility that the heat-resistant mutant xylanasecan be used in a pulp bleaching process.

With regard to c) animal feed additives, animal feed is rich in plantfibers, and plant cell walls in the animal feed can be decomposed byadding xylanase. Therefore, the efficiency of absorption of plantnutrition by animals can be improved.

In cases in which animal feed is to be pelletized using xylanase, thexylanase is required to have stability with which the xylanase cantolerate treatment at from about 70° C. to about 90° C. for about 10minutes. In addition, in order for the xylanase to work in the digestiveorgans of animals, the xylanase needs to exhibit high activity in anenvironment at about 40° C. and about pH 4.8.

Many of xylanases derived from filamentous fungi such as the genusTrichoderma and the genus Acremonium have an optimum pH of from 3 to 5and an operable temperature range around 40° C.

The heat-resistant xylanase mutants described in Patent Document 2Patent Document 3, WO 2001/27252 (Patent Document 7), and WO 2005/108565(Patent Document 8) include mutants having an optimum pH of from about 5to about 5.5.

d) Detergent Aid: The use of xylanase as a detergent aid can removefluff on clothes.

Since recent drum-type washing machines are designed to save water, finefluffing tends to occur as the number of times of washing increases.When the fluffing has occurred, re-soiling of clothes tends to occur.

The fluff can be removed by using xylanase as a detergent aid, and,therefore, re-soiling can also be prevented. Moreover, since the maincomponents of stains attaching to clothes and derived from vegetables orfruits are cell walls which are derived from the vegetables or fruitsand to which colorants are attached, effective washing can be carriedout using xylanase in washing even in cases in which a water-saving-typedrum-type washing machine is used.

In cases in which xylanase is to be used as a detergent aid, it isnecessary to use a xylanase having alkali resistance and surfactantresistance. In addition, in cases in which xylanase is used in laundrycleaning, it is necessary to use a xylanase that stably works in a hightemperature range of from 50° C. to 70° C.

A T. reesei-derived xylanase mutant having heat resistance and alkaliresistance (for examples, see Patent Document 2, Patent Document 3,Patent Document 5, and Patent Document 6) has properties including anoptimum temperature of from 62° C. to 75° C. and an optimum pH of frompH 7 to pH 8.

The xylanase mutant described in Patent Document 8 has an optimum pH ofpH 5, which is at the acidic side. However, this mutant xylanase has anoptimum temperature of 70° C., and maintains 100% activity at 60° C. andfrom pH 8 to pH 9 for at least 10 minutes.

Each of the heat-resistant and alkali-resistant xylanases derived fromthe genus Bacillus (see, for example, Patent Document 4 and JP-A No.2007-54050 (Patent Document 9)) has properties including an optimumtemperature range of from 50° C. to 70° C. and an optimum pH of from 7to 8, and maintain 100%-activity at pH 9 and from 4° C. to 5° C. for alength of time of from 1 to 2 days.

In regard to e) bread-making modifiers, the quality of bread productioncan be improved by using xylanase as a bread-making modifier.

Xylanase has properties capable of decomposing the hemicellulosecomponent of flour. Due to the decomposition of the hemicellulosecomponent by xylanase, moisture bound to this component is released intodough, thereby changing the properties of the dough. As a result, theparticle structure and the loaf volume of the produced bread areimproved, leading to favorable quality preservation of the producedbread.

When making dough, large physical impact and pressure load are appliedduring a process of stirring and kneading ingredients, and afermentation process requires a length of time of from 1 to 2 hours at atemperature of from 35° C. to 40° C.

SUMMARY OF INVENTION Problem to be Solved by Invention

However, there is still room for improvement in the reutilization of thesaccharification enzyme described in (a) saccharification oflignocellulosic raw material, from the viewpoints of the cost of sugarproduction and the effective utilization of lignocellulosic resources.

In the reutilization of the saccharification enzyme described in PatentDocument 1, it is demonstrated that the binding of the enzyme to alignocellulosic residue causes reduction in the saccharificationactivity thereof. For this reason, the addition amounts of the enzymeand the substrate are significantly limited.

In particular, the working examples of Patent Document 1 describes thatthe amount of the enzyme is an amount capable of decomposing 96% or moreof lignocellulose in 12 hours, indicating that feeding of a large amountof the enzyme is necessary. Thus, from an economical viewpoint, enzymereutilization over a long period of time is necessary.

In addition, the concentration of lignocellulose as a substrate is aslow as about 1%, and the concentration of produced sugar is also low.Therefore, for the utilization of the sugar in an ethanol fermentationprocess and the like, investment for facilities for addressing theefficiency per volume of the saccharification tank, the concentratingbefore ethanol fermentative production, and the like, is necessary.Thus, this method can hardly be regarded as an industrial method fromthe economical viewpoint.

In the saccharification of lignocellulose containing hemicellulose, itis thought that an enzyme capable of decomposing lignocellulose at highconcentration and tolerating reutilization over a long period of time isneeded. Therefore, the lowness of the stability of xylanase is aproblem, as described above.

In Patent Document 10, it is described that the activity of enzymes suchas cellulase and hemicellulase is maintained even after adsorption onresidues. Patent Document 10 also describes a method whereby asaccharification enzyme is recovered by being adsorbed on lignocelluloseafter reaction and reutilized in a next saccharification reaction.

However, in Patent Document 10, lignocellulose that will be used as asaccharification raw material is heated, in advance, under acidicconditions, whereby hemicellulose in the lignocellulose is decomposed.Therefore, heating costs are incurred, and installation of equipmentsuch as a pressure vessel having resistance to acid is needed.Accordingly, this method is not favorable from the economical viewpoint.

In view of these, decomposition of hemicellulose using xylanase isdesired. However, since the saccharification reaction is carried out fora long time, the lowness of the stability of xylanase is a problem,similar to the above.

Patent Document 11 describes that, by increasing the amount ofsaccharification enzyme to be used in an initial reaction, the amount ofenzyme to be additionally supplied, which corresponds to the activitylost at the time of reutilizing the enzyme, can be decreased, and theoverall costs for the enzyme can be decreased.

However, in fact, an amount of the enzyme additionally supplied is aslarge as ⅓ of the amount of the initially-supplied enzyme, and,therefore, this method is not favorable from the economical viewpoint.In addition, it is described, in working examples provided in PatentDocument 11, that reaction residues are disposed of. The loss of theenzyme adsorbed on the residues is a major factor that makes itimpossible to decrease the amount of the enzyme to be additionallysupplied.

Furthermore, working examples provided in Patent Document 11 describeonly the use of cellulose included in lignocellulose, namely the use ofglucose. The wheat straw used in working examples provided in PatentDocument 11, which has been subjected to pretreatment, containshemicellulose and the like in an amount of 35% or more. From theviewpoints of effective use of lignocellulose resources and economy, itis necessary to use, as a monosaccharide, xylose contained in thehemicellulose. In this case, however, the lowness of the stability ofxylanase is a problem in a situation in which the enzyme is reutilizedafter long hours of reaction that is expected to include processes fromsaccharification to ethanol fermentation.

Solutions to these may include utilization of a thermostable xylanaseprepared by improving an existing xylanase and utilization of aheat-resistant xylanase derived from a heat-resistant bacterium.However, until now, there has been no report about long-term enzymeutilization using these xylanases.

It is uncertain whether or not the T. reesei-derived heat-resistantxylanase mutants disclosed in Patent Document 2 and Patent Document 3mentioned in (a) saccharification of lignocellulosic raw materialsatisfy conditions required for the saccharification of alignocellulosic raw material (long-term use in an acidic region at hightemperatures).

Further, in regard to the heat-resistant xylanase that is derived fromthe genus Bacillus and that is described in Patent Document 4 mentionedin (a), the results of residual activity thereof upon heat treatment atpH 7.2, which is close to neutral pH, are disclosed. Therefore, theactivity of the heat-resistant xylanase is likely to decrease when theheat-resistant xylanase has been used under acidic conditions for a fewdays in order to perform the saccharification of lignocellulose.

In Patent Document 2, Patent Document 3, Patent Document 5, and PatentDocument 6, which are directed to T. reesei-derived heat-resistantxylanase mutants and mentioned in (b) pulp bleaching, data about theresidual activity of the T. reesei-derived heat-resistant xylanasesafter the T. reesei-derived heat-resistant xylanases are treated at pH 5and from 60° C. to 80° C. for 30 minutes. However, the stability of theT. reesei-derived heat-resistant xylanases under conditions simulatingpulp bleaching (at pH 10 and from 70° C. to 80° C. for a few hours) isnot demonstrated.

The xylanases derived from filamentous fungi such as the genusTrichoderma and the genus Acremonium mentioned in (c) animal feedadditives do not have thermal stability that can tolerate pelleting.

Further, among the heat-resistant xylanase mutants disclosed in PatentDocument 2, Patent Document 3, Patent Document 7, and Patent Document 8mentioned in (c), mutants having an optimum pH of from about 5 to about5.5 are included. However, all of the mutants undergo significantthermal inactivation in a high temperature range of 60° C. or higher,and, therefore, these mutants cannot be used as animal feed additives.

In regard to the T. reesei-derived heat-resistant and alkali-resistantxylanase mutants disclosed in Patent Document 2, Patent Document 3,Patent Document 5, and Patent Document 6 mentioned in (d) Detergent Aid,there is no information about the stability of the alkali-resistantxylanase mutants in a basic region over a length of time generallyrequired for washing (from 1 to 2 hours). It is thus unclear whether ornot these mutant xylanases can be used as detergent aids.

Similar to the above, it is unclear whether or not the xylanase mutantdisclosed in Patent Document 8 and the heat-resistant andalkali-resistant xylanases derived from the genus Bacillus and disclosedin Patent Document 4 and Patent Document 9, which are mentioned in (d),can tolerate the use as a detergent aid in laundry cleaning.

It is not clarified whether or not the T. reesei-derived heat-resistantxylanase mutants disclosed in Patent Document 2, Patent Document 3,Patent Document 5, and Patent Document 6, which are mentioned in (e)bread-making modifier, can tolerate large physical impact and pressureload applied during bread making.

Further, bread-making processes include a fermentation process performedat from 35° C. to 40° C. for 1 to 2 hours. Thus, compatibility with thisprocess is also required.

As described above, the range of uses in which xylanase can be used iswide. Therefore, conditions required for xylanase vary widely. Examplesthereof include severe conditions in which enzymes easily inactivate,such as a condition involving a pH of from 4 to 10, a temperature offrom 40° C. to 80° C., and a usage time of several days.

In order to address these various needs, many types of mutant xylanasesand novel xylanases have been reported. However, xylanases that can workwith sufficient stability under severe conditions in which enzymeseasily inactivate have not been found.

Mutant xylanases obtained in order to improve heat resistance have aproblem in that the initial rate of reaction largely decreases. It ispresumed that the reason therefor is a decrease in the structuralflexibility of the entire protein caused by mutations or the like of theamino acid sequence that has been introduced in order to improve heatresistance.

In such circumstances, development of a xylanase which exhibits stableactivity for a predetermined period of time under severe conditions inwhich enzymes easily inactivate such as an acidic region (from pH 4 to6), a basic region (from pH 8 to 10), or a high temperature range (from40° C. to 80° C.), and with which the initial rate of reaction is notsignificantly reduced as compared to a wild-type xylanase correspondingthereto.

The present invention aims to provide an inexpensive and efficientsaccharification method for lignocellulose using a thermostablexylanase. The invention also aims to provide a mutant xylanase that hasa substitute amino acid residue, and that exhibits stable activity evenunder severe conditions in which enzymes easily inactivate, and thatprovides an initial rate of reaction not significantly reduced ascompared to a wild-type xylanase corresponding to the mutant xylanase.The invention also aims to provide a production method capable ofproducing the mutant xylanase at low cost, as well as provide varioususes of the mutant xylanase.

Means for Solving Problem

The present invention includes the following:

[1] A method of producing a saccharified product of lignocellulose, themethod including contacting a lignocellulosic raw material with athermostable xylanase.[2] The method of producing a saccharified product according to [1],wherein the lignocellulosic raw material is pulp.[3] A method of producing a saccharified product, the method including:

recovering the thermostable xylanase from a saccharification reactionsolution containing the saccharified product of lignocellulose obtainedby the method of producing a saccharified product according to [1] or[2]; and

contacting the recovered thermostable xylanase with a lignocellulosicraw material, to produce a saccharified product.

[4] The method of producing a saccharified product according to [3],wherein the saccharification reaction solution is subjected tosolid-liquid separation using centrifugation or a microfiltrationmembrane, and the separated liquid is ultrafiltered using anultrafiltration membrane to separate and recover the saccharifiedproduct of lignocellulose and the thermostable xylanase.[5] The method of producing a saccharified product according to [4],wherein the method includes contacting a solid obtained by thesolid-liquid separation using centrifugation or a microfiltrationmembrane and the thermostable xylanase recovered using theultrafiltration membrane with a lignocellulosic raw material, to producea saccharified product.[6] The method of producing a saccharified product according to any oneof [1] to [5], wherein the thermostable xylanase is a mutant xylanasethat provides an initial rate of reaction that is at least 70% of thatprovided by a wild-type xylanase corresponding thereto, that has axylanase activity after heat treatment at 50° C. for 24 hours that is atleast 50% of its xylanase activity before the heat treatment, and thathas a substitute amino acid residue.[7] The method of producing a saccharified product according to [6],wherein the mutant xylanase is a mutant xylanase including at least thefollowing substitute amino acid residues in an amino acid sequencerepresented by SEQ ID NO: 1 in the Sequence Listing:

a leucine residue substituted for an asparagine residue at position 29;

an arginine residue substituted for a lysine residue at position 58;

an amino acid residue, other than a tyrosine residue, substituted for atyrosine residue at position 27; and

an amino acid residue, other than an asparagine residue, substituted foran asparagine residue at position 44.

[8] The method of producing a saccharified product according to [7],wherein, in the mutant xylanase used in the producing of a saccharifiedproduct, the amino acid residue, other than a tyrosine residue,substituted for the tyrosine residue at position 27 is a phenylalanineresidue, and the amino acid residue, other than an asparagine residue,substituted for an asparagine residue at position 44 is a serineresidue.[9] The method of producing a saccharified product according to [6],wherein the mutant xylanase is a mutant xylanase in which at least anamino acid residue at position 154 in the amino acid sequencerepresented by SEQ ID NO: 2 in the Sequence Listing is substituted withanother amino acid residue.[10] The method of producing a saccharified product according to [9],wherein the mutant xylanase used in the producing of a saccharifiedproduct includes at least the following substitute amino acid residues:

an aspartic acid residue substituted for an asparagine residue atposition 33;

an arginine residue substituted for a glycine residue at position 36;

a serine residue substituted for a threonine residue at position 90;

an arginine residue substituted for a glutamine residue at position 132;

a methionine residue substituted for a leucine residue at position 154;

a threonine residue substituted for a serine residue at position 174;

a histidine residue substituted for a proline residue at position 195;

an asparagine residue substituted for a serine residue at position 197;and

a glutamic acid residue substituted for a glycine residue at position217.

[11] The method of producing a saccharified product according to [9],wherein the mutant xylanase used in the producing of a saccharifiedproduct includes at least the following substitute amino acid residues:

a valine residue substituted for an isoleucine residue at position 30;

an aspartic acid residue substituted for an asparagine residue atposition 33;

an arginine residue substituted for a glycine residue at position 36;and

a methionine residue substituted for a leucine residue at position 154.

[12] The method of producing a saccharified product according to [9],wherein the mutant xylanase used in the producing of a saccharifiedproduct includes at least the following substitute amino acid residues:

a valine residue substituted for an isoleucine residue at position 30;

a threonine residue substituted for a serine residue at position 59;

a methionine residue substituted for a leucine residue at position 154;

a histidine residue substituted for a tyrosine residue at position 239;and

a serine residue substituted for a cysteine residue at position 242.

[13] A mutant xylanase including at least the following substitute aminoacid residues in an amino acid sequence represented by SEQ ID NO: 1 inthe Sequence Listing:

a leucine residue substituted for an asparagine residue at position 29;

an arginine residue substituted for a lysine residue at position 58;

an amino acid residue, other than a tyrosine residue, substituted for atyrosine residue at position 27; and

an amino acid residue, other than an asparagine residue, substituted foran asparagine residue at position 44.

[14] The mutant xylanase according to [13], wherein the amino acidresidue, other than a tyrosine residue, substituted for the tyrosineresidue at position 27 in the amino acid sequence represented by SEQ IDNO: 1 in the Sequence Listing is a phenylalanine residue, and the aminoacid residue, other than an asparagine residue, substituted for anasparagine residue at position 44 in the amino acid sequence representedby SEQ ID NO: 1 in the Sequence Listing is a serine residue.[15] A mutant xylanase including substitution of at least a leucineresidue at position 154 with another amino acid residue in the aminoacid sequence represented by SEQ ID NO: 2 in the Sequence Listing.[16] The mutant xylanase according to [15], wherein the mutant xylanaseincludes at least the following substitute amino acid residues in theamino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing:

an aspartic acid residue substituted for an asparagine residue atposition 33;

an arginine residue substituted for a glycine residue at position 36;

a serine residue substituted for a threonine residue at position 90;

an arginine residue substituted for a glutamine residue at position 132;

a methionine residue substituted for the leucine residue at position154;

a threonine residue substituted for a serine residue at position 174;

a histidine residue substituted for a proline residue at position 195;

an asparagine residue substituted for a serine residue at position 197;and

a glutamic acid residue substituted for a glycine residue at position217.

[17] The mutant xylanase according to [15], wherein the mutant xylanaseincludes at least the following substitute amino acid residues in theamino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing:

a valine residue substituted for an isoleucine residue at position 30;

an aspartic acid residue substituted for an asparagine residue atposition 33;

an arginine residue substituted for a glycine residue at position 36;and

a methionine residue substituted for the leucine residue at position154.

[18] The mutant xylanase according to [15], wherein the mutant xylanaseincludes at least the following substitute amino acid residues in theamino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing:

a valine residue substituted for an isoleucine residue at position 30;

a threonine residue substituted for a serine residue at position 59;

a methionine residue substituted for the leucine residue at position154;

a histidine residue substituted for a tyrosine residue at position 239;and

a serine residue substituted for a cysteine residue at position 242.

[19] A nucleic acid represented by a base sequence encoding the aminoacid sequence of the mutant xylanase according to any one of [13] to[18].[20] An expression vector including the nucleic acid according to [19].[21] A transformant including the expression vector according to [20].[22] The transformant according to [21], wherein a host cell of thetransformant is a cell derived from Escherichia coli, Bacillus subtilis,yeast, an actinomycete, or a filamentous fungus.[23] The transformant according to [22], wherein the filamentous fungusbelongs to the genus Trichoderma, the genus Acremonium, the genusHumicola, or the genus Aspergillus.[24] The transformant according to [22] or [23], wherein the filamentousfungus is Trichoderma viride, Acremonium cellulolyticus, Humicolainsolens, or Aspergillus niger.[25] A method of producing a mutant xylanase, the method includingculturing the transformant according to any one of [21] to [24] andrecovering the mutant xylanase according to any one of [13] to [18] fromat least one of the cultured transformant or a culture product of thetransformant.[26] A mutant xylanase produced by the production method according to[25].[27] A composition including the mutant xylanase according to any one of[13] to [18] and [21].[28] A method of bleaching a pulp, the method including contacting themutant xylanase according to any one of [13] to [18] and [21] with thepulp.[29] A detergent including the mutant xylanase according to any one of[13] to [18] and [21].[30] An animal feed including the mutant xylanase according to any oneof [13] to [18] and [21].[31] A bread-making modifier including the mutant xylanase according toany one of [13] to [18] and [21].

Advantageous Effects of Invention

According to the present invention, an inexpensive and efficientsaccharification method for lignocellulose using a thermostable xylanasecan be provided. In addition, a mutant xylanase that has a substituteamino acid residue, and that exhibits stable activity even under severeconditions in which enzymes easily inactivate, and that provides aninitial rate of reaction not significantly reduced as compared to awild-type xylanase corresponding to the mutant xylanase, can also beprovided. Furthermore, according to the invention, a production methodcapable of producing the mutant xylanase at low cost can be provided,and various uses of the mutant xylanase can also be provided.

DESCRIPTION OF EMBODIMENTS

A thermostable xylanase according to the invention may be anythermostable xylanase of which the xylanase activity after heattreatment for a specified period of time is at the same level as that ofthe xylanase activity before the heat treatment, or of which a reductionin the xylanase activity thereof after heat treatment as compared to thexylanase activity before the heat treatment is small.

Examples thereof include xylanases obtained from filamentous fungi ofthe genus Aspergillus, the genus Trichoderma, the genus Aureobasidium,the genus Schizophyllum commune, or the like, and bacteria of the genusBacillus, the genus Clostridium, and the genus Streptomyces.

Among the wild-type xylanases described above, those exhibiting axylanase activity after heat treatment at 50° C. for 24 hours that is atleast 50% of the xylanase activity thereof before heat treatment, arepreferable.

In addition, the thermostable xylanase according to the invention may bea mutant xylanase obtained by introducing a mutation into a wild-typexylanase, such as those obtained from filamentous fungi and bacteria, soas to improve thermal stability, as necessary. The mutant xylanase ismore preferably a mutant xylanase which has an initial rate of reactionthat is at least 70% of that provided by a wild-type xylanasecorresponding thereto, and of which the xylanase activity after heattreatment at 50° C. for 24 hours is at least 50% of its xylanaseactivity thereof before the heat treatment, and which includes asubstitute amino acid.

A nucleic acid according to the invention is a nucleic acid representedby a base sequence encoding an amino acid sequence of the mutantxylanase described above.

An expression vector according to the invention includes a nucleic acidrepresented by a base sequence encoding an amino acid sequence of themutant xylanase.

A host cell according to the invention is a cell which is transformedwith the expression vector including a nucleic acid represented by abase sequence encoding an amino acid sequence of the mutant xylanase.

A method of producing a mutant xylanase according to the inventionincludes culturing the host cell and collecting the mutant xylanase fromat least one of the cultured host cell or a culture product of the hostcell. The mutant xylanase according to the invention also includes amutant xylanase produced by the above-described method of producing amutant xylanase.

A composition according to the invention includes the mutant xylanase.

A method of producing a saccharified product of lignocellulose accordingto the invention includes contacting the mutant xylanase with alignocellulosic raw material.

A method of bleaching pulp according to the invention includescontacting the mutant xylanase with the pulp.

A detergent, an animal feed, or a bread-making modifier according to theinvention includes the mutant xylanase.

In the invention, descriptions about an amino acid sequence and a basesequence encoding the mutant xylanase or individual sequences of primersshall apply to the respective mentioned sequences as well as sequencescomplementary thereto, based on the mutually complementary relationshiptherebetween, unless otherwise specified. When the descriptions in theinvention are applied to the sequences complementary to the respectivesequences mentioned, the descriptions shall be interpreted, throughoutthe specification, as if sequences recognized by the complementarysequences were sequences complementary to corresponding sequencesmentioned in the present specification, within a range of commontechnical knowledge of those skilled in the art.

As used herein, the scope of the term “process” includes not only anindependent process but also a process that is not clearly distinguishedfrom other processes as long as the expected purpose of the process isachieved.

In the specification, the numerical range indicated by “(from) . . . to. . . ” indicates a range including the numerical values describedbefore and after “to” as the minimum and maximum values, respectively.

In the specification, when two or more substances, each corresponding toa particular component of a composition, are present, the amount of theparticular component in the composition means the total amount of thetwo or more substances present in the composition, unless otherwisespecified.

Hereinafter, the invention will be described.

(1) DEFINITIONS Definitions of Xylanase Activity and Initial Rate ofReaction

In the invention, the term “xylanase activity” means producing of anoligosaccharide having a reducing end (hereinafter also referred to assimply “reducing sugar”) through random hydrolysis of β-1,4 bonds ofxylan, which mainly constitutes plant cell walls.

In the invention, the term “initial rate of reaction” means an initialrate of reaction of xylanase activity.

The initial rate of reaction can be determined in the following manner.First, into 100 mM a sodium citrate buffer solution (pH 4.5), 1% (w/w)birchwood xylan (manufactured by Sigma-Aldrich Co. LLC), which is asubstrate, is vigorously mixed. Then, centrifugation at 5000×g for 15minutes was performed, to prepare a supernatant from which residualxylan present in the sodium citrate buffer solution has been removed.Next, into the supernatant as a substrate solution, xylanase is mixed inan amount of 0.1% (w/w) with respect to the substrate solution. Themixture is allowed to react while being stirred at 45° C. for 30minutes, and the amount of reducing saccharide in the obtained reactionsolution is measured by the DNS method (Bailey et al., 1992), wherebythe initial rate of reaction of the xylanase activity can be obtained.

Definition of Activity Equivalent to that of Wild-Type

As used herein, the expression “activity equivalent to that ofwild-type” means that the initial rate of reaction of a mutant xylanaseis 0.7 (70%) or higher provided that the initial rate of reaction of awild-type xylanase thereof is assumed to be 1.

Definition of Range in which Enzyme Stably Works

As used herein, the expression “range in which enzyme stably works”means a range having a temperature higher than 30° C. but lower than 40°C. and a pH larger than 6 but smaller than 8.

As used herein, the expression “severe conditions in which enzymeseasily inactivate” means an acidic region (from pH 4 to pH 6), a basicregion (from pH 8 to pH 10), and a high-temperature region (from 40° C.to 80° C.).

Definitions of Residual Activity and Stability

As used herein, the term “residual activity” refers to quotient,expressed in percentage, obtained by dividing an initial rate ofreaction after an enzyme is exposed for a certain period of time to acondition outside the range in which the enzyme stably works, by aninitial rate of reaction before the exposure. A specific measurementmethod is as follows: after heating treatment is performed at 50° C. andpH 4.5 for varied periods of 16 hours, 24 hours, 48 hours, and 72 hours,standing still on ice is performed for 5 minutes, and the initial rateof reaction is measured. The initial rate achieved by the enzyme beforethe heat treatment is also measured. Then, the division calculation isperformed and the resultant value is expressed in percentage. Inaddition to the above, residual activities are measured in the samemanner with respect to initial rates of reaction after heating treatmentis performed at 50° C. for 1 hour at pH 8, pH 9, and pH 10,respectively, initial rates of reaction after heating treatment isperformed at 60° C. for 1 hour at pH 8, pH 9, and pH 10, respectively,and an initial rate of reaction after heating treatment is performed at70° C. and pH 5.5 for 5 minutes.

In the present specification, stability is determined by the degree ofresidual activity observed when the enzyme has been exposed to severeconditions in which enzymes easily inactivate.

(2) MUTANT XYLANASE ACCORDING TO THE INVENTION

The mutant xylanase according to the invention provides an initial rateof reaction that is at least 70% of that provided by a wild-typexylanase corresponding thereto, has a xylanase activity after heattreatment at 50° C. for 24 hours that is at least 50% of its xylanaseactivity before the heat treatment, and has a substitute amino acidresidue.

Since the mutant xylanase according to the invention has a substituteamino acid residue, the mutant xylanase exhibits stable activity evenunder severe conditions in which enzymes easily inactivate, and theinitial rate of reaction thereof is not significantly reduced ascompared to a wild-type xylanase corresponding thereto.

The mutant xylanase according to the invention may be any mutantxylanase which provides an initial rate of reaction that is at least 70%of that provided by a wild-type xylanase corresponding thereto, andwhich has a xylanase activity after heat treatment at 50° C. for 24hours that is at least 50% of its xylanase activity before the heattreatment, and which has a substitute amino acid residue, and is notparticularly limited in other respects.

The mutant xylanase according to the invention preferably provides aninitial rate of reaction that is at least 70% of that provided by awild-type xylanase corresponding thereto.

When the initial rate of reaction is 70% or higher, the amount of themutant xylanase to be used does not become large as compared to theusage amount of a wild-type xylanase corresponding to the mutantxylanase, and therefore an initial rate of reaction of 70% or higher ispreferable in industrial applications.

The xylanase activity of the mutant xylanase according to the inventionafter heat treatment at 50° C. for 24 hours is preferably at least 50%,and more preferably at least 70%, of its xylanase activity before theheat treatment.

A xylanase activity after heat treatment at 50° C. for 24 hours of atleast 50% of its xylanase activity before the heat treatment ispreferable because the mutant xylanase can be used in a range in whichthe enzyme stably works. Specifically, in cases in which an enzymereaction over a long time such as saccharification of lignocellulose orreutilization of an enzyme is needed, a xylanase activity after the heattreatment that satisfies the above condition removes necessity to add alarge amount of the enzyme in order to maintain an initial rate ofreaction thereof observed at the initiation of the reaction, therebyavoiding an increase in the cost; thus, a xylanase activity after theheat treatment that satisfies the above condition is preferable alsofrom the economical viewpoint.

The origin of the mutant xylanase according to the invention is notparticularly limited. Examples of the mutant xylanase include thosederived from a Bacillus subtilis, a bacterium of the genus Clostridium,an actinomycete, a filamentous fungus, and a basidiomycete. From theviewpoint of industrial applications, mutant xylanases derived from thegenus Trichoderma, the genus Acremonium, the genus Humicola, or thegenus Aspergillus, among filamentous fungi, are preferable. From theviewpoint of mass production, mutant xylanases derived from Trichodermaviride, Acremonium cellulolyticus, Humicola insolens, or Aspergillusniger are more preferable.

More preferable mutant xylanases include the two mutant xylanasesdescribed below, from the viewpoints that stable activity is exhibitedeven under severe conditions in which enzymes easily inactivate, andthat an initial rate of reaction not significantly reduced as comparedto a wild-type xylanase corresponding thereto is provided.

A first preferable mutant xylanase is a mutant xylanase derived fromxylanase of a filamentous fungus of the genus Trichoderma.

The first preferable mutant xylanase is preferably a mutant xylanasederived from xylanase of Trichoderma viride from the viewpoints that theinitial rate of reaction is at least 70% of that provided by a wild-typexylanase corresponding thereto, and that the xylanase activity afterheat treatment at 50° C. for 24 hours is at least 50% of its xylanaseactivity before the heat treatment.

The first preferable mutant xylanase may be a mutant xylanase having, inthe amino acid sequence of SEQ ID NO: 1 in the Sequence Listing, thefollowing substitute amino acid residues: a leucine residue substitutedfor an asparagine residue at position 29; an arginine residuesubstituted for a lysine residue at position 58; an amino acid residue,other than a tyrosine residue, substituted for a tyrosine residue atposition 27; and an amino acid residue, other than an asparagineresidue, substituted for an asparagine residue at position 44. from theviewpoints of providing an initial rate of reaction that is at least 70%of that provided by a wild-type xylanase corresponding thereto, andfacilitating achievement of a xylanase activity after heat treatment at50° C. for 24 hours that is at least 50% of its xylanase activity beforethe heat treatment.

The amino acid sequence of SEQ ID NO: 1 in the Sequence Listing is anamino acid sequence encoding xylanase II of Trichoderma viride.

A second preferable mutant xylanase may be a mutant xylanase derivedfrom xylanase of a filamentous fungus belonging to the genus Acremonium.

The second preferable mutant xylanase is preferably a mutant xylanasederived from xylanase of Acremonium cellulolyticus from the viewpointsthat the initial rate of reaction is at least 70% of that provided by awild-type xylanase corresponding thereto and that the xylanase activityafter heat treatment at 50° C. for 24 hours is at least 50% of itsxylanase activity before the heat treatment.

The amino acid sequence of SEQ ID NO: 2 in the Sequence Listing is anamino acid sequence encoding xylanase I of Acremonium cellulolyticus.

The second preferable mutant xylanase preferably includes at least asubstitute amino acid residue at position 154 that is a methionineresidue substituted for a leucine residue, from the viewpoint offacilitating achievement of an initial rate of reaction of at least 70%of that provided by a wild-type xylanase corresponding thereto, andachievement of a xylanase activity after heat treatment at 50° C. for 24hours that is at least 50% of its xylanase activity before the heattreatment.

Specific examples of the mutant xylanase according to the inventioninclude those having a substitute amino acid residue and represented byclone Nos. 1 to 17 shown in Table 1. However, the mutant xylanaseaccording to the invention is not limited thereto.

TABLE 1 Clone Sequence No. Position of Substitute Before After No.(Wild-Type) Amino Acid Residue Mutation Mutation 1 1 27 Tyr Phe 1 29 AsnLeu 1 44 Asn Ser 1 58 Lys Arg 2 2 30 Ile Val 3 2 33 Asn Asp 4 2 36 GlyArg 5 2 59 Ser Thr 6 2 90 Thr Ser 7 2 132 Gln Arg 8 2 154 Leu Met 9 2174 Ser Thr 10 2 195 Pro His 11 2 197 Ser Asn 12 2 217 Gly Glu 13 2 239Tyr His 14 2 242 Cys Ser 15 2 33 Asn Asp 2 36 Gly Arg 2 90 Thr Ser 2 132Gln Arg 2 154 Leu Met 2 174 Ser Thr 2 195 Pro His 2 197 Ser Asn 2 217Gly Glu 16 2 30 Ile Val 2 33 Asn Asp 2 36 Gly Arg 2 154 Leu Met 17 2 30Ile Val 2 59 Ser Thr 2 154 Leu Met 2 239 Tyr His 2 242 Cys Ser

In Table 1, from the viewpoint of providing an initial rate of reactionthat is at least 70% of that provided by a wild-type xylanasecorresponding thereto and a xylanase activity after heat treatment at50° C. for 24 hours that is at least 50% of its xylanase activity beforethe heat treatment, a mutant xylanase TVX01 (Clone No. 1), a mutantxylanase ACX 01 (Clone No. 15), a mutant xylanase ACX02 (Clone No. 16),or a mutant xylanase ACX03 (Clone No. 17) is preferable.

The mutant xylanase TVX01 includes the following substitute amino acidresidues incorporated into the amino acid sequence of SEQ ID NO: 1 inthe Sequence Listing: a leucine residue substituted for an asparagineresidue at position 29, an arginine residue substituted for a lysineresidue at position 58, a phenylalanine residue substituted for atyrosine residue at position 27, and a serine residue substituted for anasparagine residue at position 44. The mutant xylanase TVX01 ispreferable from the viewpoints of providing an initial rate of reactionthat is at least 70% of that provided by a wild-type xylanasecorresponding thereto and a xylanase activity after heat treatment at50° C. for 24 hours that is at least 50% of its xylanase activity beforethe heat treatment.

The mutant xylanase TVX 01 according to the invention has activity in arange of preferably from 30° C. to 90° C., and more preferably from 30°C. to 70° C. In addition, the mutant xylanase TVX01 has activity in arange of preferably from pH 3 to 9, and more preferably from pH 4 to 7.

The mutant xylanase ACX01 includes a substitute amino acid residue thatis an aspartic acid residue substituted for an asparagine residue atposition 33, a substitute amino acid residue that is an arginine residuesubstituted for a glycine residue at position 36, a substitute aminoacid residue that is a serine residue substituted for a threonineresidue at position 90, a substitute amino acid residue that is anarginine residue substituted for a glutamine residue at position 132, asubstitute amino acid residue that is a methionine residue substitutedfor a leucine residue at position 154, a substitute amino acid residuethat is a threonine residue substituted for a serine residue at position174, a substitute amino acid residue that is a histidine residuesubstituted for a proline residue at position 195, a substitute aminoacid residue that is an asparagine residue substituted for a serineresidue at position 197, and a substitute amino acid residue that is aglutamic acid residue substituted for a glycine residue at position 217.The mutant xylanase ACX01 is preferable from the viewpoints of aninitial rate of reaction that is at least 70% of that provided by awild-type xylanase corresponding thereto and a xylanase activity afterheat treatment at 50° C. for 24 hours that is at least 50% of itsxylanase activity before the heat treatment.

The mutant xylanase ACX01 according to the invention has activity in arange of preferably from 30° C. to 80° C., and more preferably 30° C. to65° C. In addition, the mutant xylanase ACX01 has activity in a range ofpreferably from pH 2 to 8, and more preferably from pH 2 to 5.

The mutant xylanase ACX02 includes a substitute amino acid residue thatis a valine residue substituted for an isoleucine residue at position30, a substitute amino acid residue that is an aspartic acid residuesubstituted for an asparagine residue at position 33, a substitute aminoacid residue that is an arginine residue substituted for a glycineresidue at position 36, and a substitute amino acid residue that is amethionine residue substituted for a leucine residue at position 154.The mutant xylanase ACX02 is preferable from the viewpoints of providingan initial rate of reaction that is at least 70% of that provided by awild-type xylanase corresponding thereto and a xylanase activity afterheat treatment at 50° C. for 24 hours that is at least 50% of itsxylanase activity before the heat treatment.

The mutant xylanase ACX02 according to the invention has activity in arange of preferably from 30° C. to 80° C., and more preferably from 30°C. to 65° C. In addition, the mutant xylanase ACX02 has activity in arange of preferably from pH 2 to 8, and more preferably from pH 2 to 5.

The mutant xylanase ACX03 includes a substitute amino acid residue thatis a valine residue substituted for an isoleucine residue at position30, a substitute amino acid residue that is a threonine residuesubstituted for a serine residue at position 59, a substitute amino acidresidue that is a methionine residue substituted for a leucine residueat position 154, a substitute amino acid residue that is a histidineresidue substituted for a tyrosine residue at position 239, and asubstitute amino acid residue that is a serine residue substituted for acysteine residue at position 242. The mutant xylanase ACX03 ispreferable from the viewpoints of providing an initial rate of reactionthat is at least 70% of that provided by a wild-type xylanasecorresponding thereto and a xylanase activity after heat treatment at50° C. for 24 hours that is at least 50% of its xylanase activity beforethe heat treatment.

The mutant xylanase ACX03 according to the invention has activity in arange of preferably from 30° C. to 80° C., and more preferably from 30°C. to 65° C. In addition, the mutant xylanase ACX03 has activity in arange of preferably from pH 2 to 8, and more preferably from pH 2 to 5.

The scope of the mutant xylanase according to the invention alsoincludes mutant xylanases consisting of amino acid sequences homologousto the mutant xylanase TVX01.

The “amino acid sequences homologous thereto” may be, for example, aminoacid sequences that exhibit approximately equivalent level of xylanaseactivity as that of the mutant xylanase TVX01. Preferable examplesinclude mutant xylanases having a homology of 80% or higher, morepreferably 90% or higher, and still more preferably 95% or higher, withthe amino acid sequence of the mutant xylanase TVX01. A homology of 80%or higher is considered to provide a higher similarity between thesteric structures of the xylanases, thereby providing an advantage inthat, for example, a mutant xylanase exhibiting approximately equivalentlevel of activity as that according to the invention can be developed byintroducing one or more mutation sites clarified by the invention.

The same applies to the mutant xylanases ACX01, ACX02, and ACX03, inaddition to the mutant xylanase TVX01.

The scope of the mutant xylanase TVX01 according to the invention alsoencompasses mutant xylanases in which insertion, deletion, orsubstitution of one or more amino acid residues has been introduced intothe amino acid sequence encoding the mutant xylanase TVX01, and whichexhibit approximately equivalent level of activity as that of the mutantxylanase TVX01.

In cases in which one or more amino acid residues are inserted, deleted,or substituted, the position(s) of the insertion, the deletion, or thesubstitution may be freely selected as long as the effects exerted bythe invention are not impaired. The number of amino acid residues thatare inserted, deleted, or substituted may be one amino acid residue, ortwo or more amino acid residues, for example, from one amino acidresidue to ten amino acid residues, preferably from one amino acidresidue to five amino acid residues. Specific examples include: a mutantxylanase in which the mutations at the four sites as well assubstitution of a glycine residue at position 47 with a cysteine residuehave been introduced into the amino acid sequence of SEQ ID NO: 1 in theSequence Listing; a mutant xylanase in which the mutations at the foursites as well as substitution of a glutamine residue at position 52 witha lysine residue have been introduced into the amino acid sequence ofSEQ ID NO: 1 in the Sequence Listing; a mutant xylanase in which themutations at the four sites as well as substitution of an valine residueat position 59 with an isoleucine residue have been introduced into theamino acid sequence of SEQ ID NO: 1 in the Sequence Listing; a mutantxylanase in which the mutations at the four sites as well assubstitution of an asparagine residue at position 67 with an asparticacid residue have been introduced into the amino acid sequence of SEQ IDNO:1 in the Sequence Listing; a mutant xylanase in which the mutationsat the four sites as well as substitution of an asparagine residue atposition 69 with an isoleucine residue have been introduced into theamino acid sequence of SEQ ID NO: 1 in the Sequence Listing; a mutantxylanase in which the mutations at the four sites as well assubstitution of an serine residue at position 80 with an alanine residuehave been introduced into the amino acid sequence of SEQ ID NO: 1 in theSequence Listing; a mutant xylanase in which the mutations at the foursites as well as substitution of an asparagine residue at position 97with an aspartic acid residue have been introduced into the amino acidsequence of SEQ ID NO: 1 in the Sequence Listing; a mutant xylanase inwhich the mutations at the four sites as well as substitution of aleucine residue at position 105 with a methionine residue have beenintroduced into the amino acid sequence of SEQ ID NO: 1 in the SequenceListing; a mutant xylanase in which the mutations at the four sites aswell as substitution of an threonine residue at position 109 with analanine residue have been introduced into the amino acid sequence of SEQID NO: 1 in the Sequence Listing; a mutant xylanase in which themutations at the four sites as well as substitution of an threonineresidue at position 120 with an arginine residue have been introducedinto the amino acid sequence of SEQ ID NO: 1 in the Sequence Listing; amutant xylanase in which the mutations at the four sites as well assubstitution of an threonine residue at position 143 with an isoleucineresidue have been introduced into the amino acid sequence of SEQ ID NO:1 in the Sequence Listing; a mutant xylanase in which the mutations atthe four sites as well as substitution of an asparagine residue atposition 151 with a serine residue have been introduced into the aminoacid sequence of SEQ ID NO: 1 in the Sequence Listing; a mutant xylanasein which the mutations at the four sites as well as substitution of aserine residue at position 161 with a leucine residue have beenintroduced into the amino acid sequence of SEQ ID NO: 1 in the SequenceListing; and a mutant xylanase in which the mutations at the four sitesas well as substitution of a serine residue at position 186 with athreonine residue have been introduced into the amino acid sequence ofSEQ ID NO: 1 in the Sequence Listing.

The same applies to the mutant xylanases ACX01, ACX02, and ACX03, inaddition to the mutant xylanase TVX01. Specific examples include amutant xylanase in which the mutation sites defined in the ACX01 as wellas substitution of a serine residue at position 133 with an asparagineresidue have been introduced into the amino acid sequence of SEQ ID NO:2 in the Sequence Listing, and a mutant xylanase in which the mutationsites defined in the ACX01 as well as substitution of a glutamineresidue at position 176 with an arginine residue have been introducedinto the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing.

Similarly, regarding the mutant xylanase ACX02, many mutants having allof the mutation sites defined in the ACX02 exhibit propertiesapproximately equivalent to those of the ACX02. Specific examplesthereof include: a mutant xylanase in which the mutation sites definedin ACX02 as well as substitution of a threonine residue at position 90with a serine residue have been introduced into the amino acid sequenceof SEQ ID: NO: 2 in the Sequence Listing; a mutant xylanase in which themutation sites defined in ACX02 as well as substitution of a glutamineresidue at position 132 with an arginine residue have been introducedinto the amino acid sequence of SEQ ID: NO: 2 in the Sequence Listing; amutant xylanase in which the mutation sites defined in ACX02 as well assubstitution of a serine residue at position 133 with an asparagineresidue have been introduced into the amino acid sequence of SEQ ID: NO:2 in the Sequence Listing; a mutant xylanase in which the mutation sitesdefined in ACX02 as well as substitution of a serine residue at position174 with a threonine residue have been introduced into the amino acidsequence of SEQ ID: NO: 2 in the Sequence Listing; a mutant xylanase inwhich the mutation sites defined in ACX02 as well as substitution of aproline residue at position 195 with a histidine residue have beenintroduced into the amino acid sequence of SEQ ID: NO: 2 in the SequenceListing; a mutant xylanase in which the mutation sites defined in ACX02as well as substitution of a glutamine residue at position 176 with anarginine residue have been introduced into the amino acid sequence ofSEQ ID: NO: 2 in the Sequence Listing; a mutant xylanase in which themutation sites defined in ACX02 as well as substitution of a serineresidue at position 197 with an asparagine residue have been introducedinto the amino acid sequence of SEQ ID: NO: 2 in the Sequence Listing;and a mutant xylanase in which the mutation sites defined in ACX02 aswell as substitution of a glycine residue at position 217 with aglutamic acid residue have been introduced into the amino acid sequenceof SEQ ID: NO: 2 in the Sequence Listing.

Furthermore, many mutants having all of the mutation sites defined inthe mutant xylanase ACX03 exhibit properties approximately equivalent tothose of the ACX03. Specific examples thereof include a mutant xylanasein which the mutation sites defined in ACX03 as well as substitution ofa glutamine residue at position 176 with an arginine residue have beenintroduced into the amino acid sequence of SEQ ID: NO: 2 in the SequenceListing.

The mutant xylanase according to the invention can be synthesizedaccording to known methods. Examples of a method for generating amutation in a gene include site-directed mutagenesis (Kramer, W. andfrita, H. J., Methods in Enzymology, vol. 154, P. 350 (1987)),recombinant PCR (PCR Technology, Stockton Press (1989)), chemicalsynthesis of DNA of a specific site, hydroxylamine treatment of thegene, and a method including treating a microorganism having the genewith UV irradiation or a chemical agent such as nitrosoguanidine ornitrous acid. Among methods for obtaining the mutant xylanase accordingto the invention, preferable methods include the method of producing amutant xylanase described below.

(3) METHOD OF PRODUCING MUTANT XYLANASE

A method of producing a mutant xylanase according to the invention(hereinafter referred to as simply “production method”) includesculturing a transformant and recovering the mutant xylanase from atleast one of the cultured transformant or a culture product of thetransformant.

Here, the term “transformant” refers to a transformant transformed withan expression vector that includes a nucleic acid represented by a basesequence encoding the amino acid sequence of the mutant xylanase.

In a method of producing a mutant xylanase according to the inventionincludes culturing a transformant transformed with an expression vectorthat includes a nucleic acid represented by a base sequence encoding theamino acid sequence of the mutant xylanase, to produce the mutantxylanase. With this production method, a mutant xylanase that exhibitsstable activity even under severe conditions in which enzymes easilyinactivate, and that provides an initial rate of reaction notsignificantly reduced as compared to a wild-type xylanase correspondingthereto, can be produced at low cost.

Processes that may be included in the production method are describedbelow. The method of producing a mutant xylanase according to theinvention includes a process of culturing a transformant transformedwith an expression vector that includes a nucleic acid represented by abase sequence encoding the amino acid sequence of the mutant xylanase (ahost cell cultivation process) and a process of recovering the mutantxylanase from at least one of the cultured transformant or a cultureproduct of the transformant (a mutant xylanase recovery process). Themethod of producing a mutant xylanase according to the invention mayfurther include other processes, as necessary.

A. Transformant Cultivation Process

The transformant cultivation process is a process of culturing atransformant transformed with an expression vector that includes anucleic acid represented by a base sequence encoding the amino acidsequence of the mutant xylanase.

[Transformant]

In the production method according to the invention, the transformant istransformed with an expression vector that includes a nucleic acidrepresented by a base sequence encoding the amino acid sequence of themutant xylanase, and the transformant is not particularly limited inother respects.

Examples of the transformant include host cells derived from Escherichiacoli, Bacillus subtilis, yeasts, actinomycetes, filamentous fungi, orthe like. Among them, transformants of which the host cells are derivedfrom Bacillus subtilis, yeasts, actinomycetes, or filamentous fungi,each enabling production of the target enzyme by secretion to outsidetheir cells, are preferable from the viewpoint of industrialapplications.

Examples of the yeasts include those belonging to the genusSaccharomyces, the genus Hansenula, or the genus Pichia. One example ofpreferable yeasts is Saccharomyces cerevisiae.

Examples of the filamentous fungi include those belonging to the genusHumicola, the genus Aspergillus, the genus Trichoderma, or the genusAcremonium. Preferable examples of the filamentous fungi are Humicolainsolens, Aspergillus niger, Aspergillus oryzae, Trichoderma viride, orAcremonium cellulolyticus. From the viewpoint of industrialapplications, Trichoderma viride, Acremonium cellulolyticus, Humicolainsolens, or Aspergillus niger is more preferable.

[Nucleic Acid]

The nucleic acid described above is represented by a base sequenceencoding the amino acid sequence of the mutant xylanase.

Examples of methods for synthesizing the base sequence encoding theamino acid sequence of the mutant xylanase include a method ofintroducing one or more mutation sites into a base sequence encoding acorresponding wild-type xylanase, and a method of chemicallysynthesizing the entire base sequence that includes one or more mutationsites. The method of introducing one or more mutation sites into a basesequence encoding a corresponding wild-type xylanase is described belowusing a base sequence encoding a xylanase I of Acremonium cellulolyticusand a base sequence encoding a xylanase II of Trichoderma viride.However, the nucleic acid according to the invention is not limitedthereto.

[Introduction of Mutation Sites into Base Sequence Encoding Wild-TypeXylanase]

Examples of the base sequences encoding the wild-type xylanases includea base sequence encoding the xylanase I of Acremonium cellulolyticusrepresented by SEQ ID NO: 3 in the Sequence Listing and a base sequenceencoding the xylanase II of Trichoderma viride represented by SEQ ID NO:4 in the Sequence Listing.

Examples of a method of generating a mutation in a gene using a basesequence encoding a wild-type xylanase such as those mentioned above asa template include a site-directed mutagenesis method (Kramer, W. andfrita H. J., Methods in Enzymology, vol. 154, P. 350 (1987)), arecombinant PCR method (PCR Technology, Stockton Press (1989)), a methodof chemically synthesizing a particular portion of a DNA, a method oftreating a gene with hydroxylamine, a method of subjecting amicroorganism possessing the gene to UV irradiation treatment or totreatment with a chemical agent such as nitrosoguanidine or nitrousacid, and commercially available kits for introducing mutations. Amutation can be introduced into the base sequence using these methods.

The positions and types of introduced mutations are not particularlylimited. The mutation sites of the clones represented by Clone Nos. 1 to17 are indicated as specific examples in Table 2 below. However, thepositions and types of introduced mutations are not limited thereto.

TABLE 2 SEQ ID NO: Positions  Before Clone No. (Wild-type) of BasesMutation After Mutation  1 1  79 to 81 TAC TTC, TTT 1  85 to 87 AATCTC, TTA, TTG, CTT, CTA, CTG 1 130 to 132 AACAGC, TCT, TCC, TCA, TCG, AGT 1 172 to 174 AAGAGG, CTG, CGC, CGA, CGG, AGA  2 2 88 to 90 ATC GTC, GTT, GTA, GTG  3 297 to 99 AAT GAT, GAC  4 2 106 to 108 GGG AGG, CTG, CGA, CGC, CGG, AGA 5 2 175 to 177 TCG ACG, ACT, ACC, ACA  6 2 268 to 270 ACTTCT, TCC, TCA, TCG, AGT, AGC  7 2 394 to 396 CAACGA, CTG, CGC, CGG, AGA, AGG  8 2 460 to 462 TTG ATG  9 2 520 to 522 TCTACT, ACC, ACA, ACG 10 2 583 to 585 CCC CAC, CAT 11 2 589 to 591 AGCAAC, AAT 12 2 649 to 651 GGA GAA, GAG 13 2 715 to 717 TAC CAC, CAT 14 2724 to 726 AGC AGC, TCT, TCC, TCA, TCG, AGT 15 2  97 to 99 AAT GAT, GAC2 106 to 108 GGG AGG, CTG, CGC, CGA, CGG, AGA 2 268 to 270 ACTTCT, TCC, TCA, TCG, AGT, AGC 2 394 to 396 CAACGA, CTG, CGC, CGG, AGA, AGG 2 460 to 462 TTG ATG 2 520 to 522 TCTACT, ACC, ACA, ACG 2 583 to 585 CCC CAC, CAT 2 589 to 591 AGC AAC, AAT 2649 to 651 GGA GAA, GAG 16 2  88 to 90 ATC GTC, GTT, GTA, GTG 2 97 to 99 AAT GAT, GAC 2 106 to 108 GGG AGG, CTG, CGA, CGC, CGG, AGA 2460 to 462 TTG ATG 17 2  88 to 90 ATC GTC, GTT, GTA, GTG 2 175 to 177TCG ACG, ACT, ACC, ACA 2 460 to 462 TTG ATG 2 715 to 717 TAC CAC, CAT 2724 to 726 AGC AGC, TCT, TCC, TCA, TCG, AGT

[Expression Vector]

The expression vector includes a nucleic acid represented by a basesequence encoding the amino acid sequence of the mutant xylanase, and isnot particularly limited in other respects. From the viewpoint ofimproving transformation efficiency or translation efficiency, theexpression vector is more preferably a plasmid vector or a phage vector,each of which has a structure as discussed below.

[Basic Structure of Expression Vector]

The expression vector includes a base sequence encoding the mutantxylanase and is capable of transforming the host cell, and theexpression vector is not particularly limited in other respects. Inaddition to the base sequence described above, the expression vector mayfurther include a base sequence that constitutes another region(hereinafter referred to as simply “another region”), if necessary.

Examples of the another region include a control region necessary forthe transformant to produce the mutant xylanase and a region necessaryfor autonomous replication.

From the viewpoint of facilitating the selection of the transformant,the expression vector may further include a base sequence encoding agene for selection that can serve as a selection marker.

Examples of the control region necessary to produce the mutant xylanaseinclude a promoter sequence (including an operator sequence thatcontrols transcription), a ribosome binding sequence (SD sequence), anda transcription terminator sequence.

[Expression Vector in Case where Host Cell is Yeast]

In cases in which yeast is used as a host cell, the expression vectorpreferably includes a promoter sequence in addition to the base sequenceencoding the mutant xylanase, from the viewpoint of efficiency ofproduction of the mutant xylanase. The promoter sequence may be anysequence that allows the expression of the mutant xylanase in atransformant of which the host cell is yeast.

For example, promoter sequences, such as an alcohol dehydrogenase (ADH1)promoter, a phosphoglycerate kinase (PGK1) promoter, a peptide chainelongation factor (TEF) promoter, a glycerol-3-phosphate dehydrogenase(GPD) promoter, a galactokinase (GAL1) promoter, a metallothionein(CUP1) promoter, a repressible acid phosphatase (PHO5) promoter, and aglyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, are employed.

The origins of the promoter sequences are not limited to the yeast,which serves as the host cell.

Exogenous promoters such as a cytomegalovirus (CMV) promoter may beused. These promoters may be selected, as appropriate, in accordancewith the origin and type of the enzyme to be used.

The expression vector may also include a secretion signal. Inclusion ofthe secretion signal allows the mutation xylanase to be secreted tooutside the cell when the transformant has produced the mutant xylanase.

The secretion signal should allow the mutant xylanase to be secretedfrom the yeast serving as the host cell, and is not particularly limitedin other respects. From the viewpoint of secretion efficiency, it ispreferable to use an a factor signal sequence, an invertase signalsequence, an acid phosphatase signal sequence, a glucoamylase signalsequence, or the like.

Specific examples of expression vectors that include a promoter sequenceor a secretion signal, such as those described above, include pRS423,pRS424, and YEplac195.

[Expression Vector in Case where Host Cell is Filamentous Fungus]

In cases in which a filamentous fungus is used as a host cell, theexpression vector preferably includes a promoter sequence in addition tothe base sequence encoding the mutant xylanase, from the viewpoint ofthe efficiency of production of the mutant xylanase. The promotersequence may be any sequence that allows the expression of the mutantxylanase in a transformant of which the host cell is a filamentousfungus.

Expression vectors suitable for filamentous fungi are described in vanden Hondel, C. A. M. J. J. et al. (1991) In: Bennett, J. W. and Lasure,L. L. (eds.) More gene Manipulations in Fungi. Academic Press, pp.396-428.

In addition, other commonly used expression vectors are also usable,such as pUC18, pBR322, pUC100, pSL1180 (manufactured by Pharmacia,Inc.), pFB6, Aspergillus pRAX, and Trichoderma pTEX.

[Expression Vector in Case where Host Cell is Prokaryote]

In cases in which the host cell is a prokaryote such as Escherichiacoli, Bacillus subtilis, or an actinomycete, the expression vectorpreferably includes a promoter sequence in addition to the base sequenceencoding the mutant xylanase, from the viewpoint of the efficiency ofproduction of the mutant xylanase. Besides the promoter sequence, theexpression vector may include a ribosome binding sequence, atranscription terminator sequence, or the like.

Examples of the promoter sequence include a tryptophan operon (trp)promoter and a lactose operon (lac) promoter, which are derived fromEscherichia coli, a PL promoter and a PR promoter, which are derivedfrom lambda phage, a gluconic acid synthetase promoter (gnt), analkaline protease promoter (apr), a neutral protease promoter (npr), andan α-amylase promoter (amy), which are derived from Bacillus subtilis.

Independently modified or designed promoter sequences, such as a tacpromoter, is also usable.

The ribosome binding sequence may be a sequence derived from Escherichiacoli or Bacillus subtilis. The ribosome binding sequence should functionin a desired host cell, such as in Escherichia coli or Bacillussubtilis, but is not particularly limited in other respects.

Examples of the ribosome binding sequence include a consensus sequencethat consists of four or more consecutive bases in a sequencecomplementary to the 3′ end region of 16S ribosome RNA and that has beenproduced by DNA synthesis.

The transcription terminator sequence is not essential. Transcriptionterminator sequences that are not dependent on p factor, such as alipoprotein terminator and a trp operon terminator, may be used.

The order in which these control regions are arranged in the expressionvector is not particularly limited. In consideration of transcriptionefficiency, it is preferable that a promoter sequence, a ribosomebinding sequence, a gene encoding a target protein, and a transcriptionterminator sequence are arranged in this order from the upstream at the5′-terminal side.

In regard to specific examples of expression vectors as used herein,pBR322, pUC18, Bluescript II SK(+), pKK223-3, and pSC101, which have aregion capable of autonomously replicating in Escherichia coli, andpUB110, pTZ4, pC194, ρ11, φ1, and φ105, which have a region capable ofautonomously replicating in Bacillus subtilis, may be utilized as theexpression vectors.

In addition, in regard to examples of expression vectors capable ofautonomous replication in two or more types of host cells, pHV14, TRp7,YEp7, pBS7, and the like may be used as the expression vectors.

[Method of Producing Transformant]

The transformant according to the invention can be produced by knownmethods. Examples thereof include a method including constructing anexpression vector that includes a base sequence encoding the mutantxylanase according to the invention and that optionally includes theanother region, and transforming a desired host cell with the expressionvector. Specifically, general methods known in the fields of molecularbiology, bioengineering, and genetic engineering may be employed, suchas those described in Sambrook, J., et. al., “Molecular Cloning ALaboratory Manual, 3rd Edition”, Cold Spring Harbor Laboratory Press,(2001).

The transformant according to the invention may be produced by, forexample, incorporating a silent mutation such that a codon having lowfrequency of use in the host cell is replaced by a codon having highfrequency of use in the host cell, in accordance with the necessity, inaddition to incorporating the expression vector into the host cell.

There is a possibility that the production amount of the protein derivedfrom the mutant xylanase incorporated in the expression vector isincreased thereby.

Table 3 below illustrates an example of the manners in which silentmutations are introduced. Methods for introduction of silent mutationsare not particularly limited with respect to technique, mutation sites,types of bases to be changed, and the like as long as the methods enablemodification of the codons of the xylanase gene in the expression vectorand the codons of the signal sequence for causing secretion of thexylanase gene to the outside of the cell, based on the usage frequenciesof the codons in the host cell.

Table 3 below indicates the base positions at which silent mutations areadded in order to allow expression of the mutation xylanase ACX02 athigh frequency in T. viride, and the types of the bases to be changed.

In Table 3, the “base positions” for the sequence name “ACX02” indicatethe positions of bases in SEQ ID NO: 4. The “base positions” for thesequence name “A. cellulolyticus signal sequence” indicates thepositions of bases in SEQ ID NO: 73.

TABLE 3 Base Before After Sequence Name Positions Change Change A.cellulolyticus 12 A C signal sequence 42 G T 66 A C 90 G C ACX02 37 A T38 G C 39 T C 78 T A 81 T C 106 A C 108 G C 138 A C 279 A C 312 A C 405G C 474 A C 495 A C 552 T C 573 A C 648 G A 663 C G 718 A T 719 G C 720T C

[Method of Culturing Transformant]

Conditions for culturing a transformant obtained by transformation withthe expression vector are as described in the explanation of theconditions for culturing a host cell before transformation, and knownconditions may be used.

In regard to the culture medium, both of a synthesized medium or anatural medium are usable, provided that the medium contains a carbonsource, a nitrogen source, an inorganic substance, and other nutrientsin appropriate amounts. Known components for culture media may beemployed. For example, organic nutritional sources such as meat extract,yeast extract, malt extract, peptone, NZ amine, and potatoes, carbonsources such as glucose, maltose, sucrose, starch, and organic acids,nitrogen sources such as ammonium sulfate, urea, and ammonium chloride,inorganic nutrient sources such as phosphate salts, magnesium,potassium, and iron, and vitamins, may be used in appropriatecombinations.

In the cultivation of a transformant transformed with the expressionvector that includes a selection marker, for example, in cases in whichthe selection marker is a drug-resistant selection marker, a medium thatcontains a drug corresponding to the drug-resistant selection marker isused, whereas, in cases in which the selection marker is an auxotrophicselection marker, a medium that does not contain a nutrientcorresponding to the auxotrophic selection marker is used. The pH of themedium may be selected within a range of from pH 4 to pH 8.

The cultivation may be performed by culturing the transformant in aliquid medium that contains the medium described above, using anordinary culture method such as shaking culture, aeration-stirringculture, continuous culture, or fed-batch culture.

Culture conditions may be selected, as appropriate, in accordance withthe type of transformant, the type of medium, and the type of culturemethod. The culture conditions should enable the transformant to growand produce the mutant xylanase according to the invention, and theculture conditions are not particularly limited in other respects.

The culture temperature is from 20° C. to 45° C., and preferably from24° C. to 37° C., and the cultivation is performed aerobically.

The culture period may be set to a period in the range of from 1 day to7 days, and the cultivation may be continued until the content of theprotein having the desired mutant xylanase activity reaches the maximum.

B. Mutant Xylanase Recovery Process

The mutant xylanase recovery process is a process of recovering themutant xylanase from at least one of the cultured transformant or aculture product of the transformant.

The method for recovering the mutant xylanase according to the inventionafter culturing the transformant obtained by transformation may be amethod commonly used in the art.

In cases in which the mutant xylanase according to the invention issecreted to outside the transformant obtained by transformation, a crudeenzyme solution can be easily obtained by subjecting the culture productof the transformant to centrifugation, filtration, or the like. In casesin which the mutant xylanase according to the invention is accumulatedin the transformant obtained by transformation, a crude enzyme solutionmay be recovered by recovering the cultured transformant using a meanssuch as centrifugation, suspending the recovered transformant in abuffer solution, and breaking the cell membrane of the transformantusing a known method such as lysozyme treatment, freezing and thawing,or ultrasonic disintegration.

The crude enzyme solution can be used as a concentrated enzyme by beingconcentrated by an ultrafiltration method or the like and supplementedwith a preservative or the like. A powder enzyme of the mutant xylanasecan be obtained by using, for example, a spray-drying method after theconcentration.

In cases in which the recovered crude enzyme solution having a xylanaseactivity needs to be separated and purified, for example, salting-outusing ammonium sulfate or the like, organic solvent precipitationmethods using alcohol or the like, membrane separation methods usingdialysis, ultrafiltration or the like, and known chromatographicseparation methods such as ion-exchanger chromatography, reversed-phasehigh-speed chromatography, affinity chromatography, and gel filtrationchromatography, may be performed in appropriate combinations.

(4) USE OF MUTANT XYLANASE

As described above, the mutant xylanase according to the invention hasstable activity over a long period of time even under conditions inwhich enzymes easily inactivate. Therefore, the mutant xylanaseaccording to the invention can be used in a wide range of uses.

A composition according to the invention includes the mutant xylanasedescribed above, and may also include freely-selected componentssuitable for the desired application, if necessary.

The composition according to the invention includes the mutant xylanasedescribed above, which works stably over a long period of time evenunder conditions in which enzymes easily inactivate. Therefore, thecomposition according to the invention can be used for various uses.

The content of the mutant xylanase may be set, as appropriate, inaccordance with the use of the composition, and is not particularlylimited.

The mutant xylanase according to the invention can be used in varioususes. The mutant xylanase is preferably utilized in the manner describedbelow.

[Method of Producing Saccharide from Lignocellulosic Raw Material]

The method of producing a saccharified product of lignocelluloseaccording to the invention includes contacting the mutant xylanase witha lignocellulosic raw material.

In the method of producing a saccharified product of lignocelluloseaccording to the invention, the mutant xylanase described above, whichis able to work stably over a long period of time even under a conditionin which enzymes easily inactivate, is used; therefore, the productioncan be performed under conditions in which enzymes easily inactivate,and saccharification of lignocellulose can be efficiently achieved.

Known lignocellulosic raw materials having a low lignin content may beused as the lignocellulosic raw material.

The phrase “low lignin content” refers to a lignin content of lower than30% by mass, considering that the average lignin content oflignocellulosic raw materials is about 30% by mass with respect to thetotal amount of lignocellulosic raw material. Lignocellulosic rawmaterials having a lignin content of 20% by mass or lower arepreferable, and lignocellulosic raw materials having a lignin content of10% by mass or lower are more preferable.

Examples of the lignocellulosic raw material include pulp fibers thatinclude cellulose and hemicellulose as main components, and that areobtained by high-degree removal of lignin from lignocellulosic materialssuch as softwood, hardwood, a logging residue, construction waste wood,pruning waste, sawdust, kenaf, and agricultural wastes such as ricestraw and wheat straw using a chemical pulp production method such asalkali extraction or alkaline digestion or using a method such asorganosolve. Preferable examples thereof include hardwood kraft pulp,softwood kraft pulp, mechanical pulp, pulp derived from a herbaceousplant such as kenaf, wastepaper or paper sludge (including pulp fibercontent recovered from a paper pulp mill), or any mixture thereof. Inparticular, hardwood kraft pulp and softwood kraft pulp are morepreferable.

Each of these lignocellulosic raw materials is available from generalpulp manufacturing companies.

Examples of methods for contacting the mutant xylanase with alignocellulosic raw material include: a method including adding themutant xylanase to the lignocellulosic raw material and allowing thereaction to proceed while stirring; a method including allowing thereaction to proceed while shaking; and a method including sufficientlymixing the mutant xylanase and the lignocellulose and then allowing themixture to stand still so as to allow the reaction to proceed. From theviewpoint of reaction efficiency, a preferable method is a methodincluding adding the mutant xylanase to a lignocellulosic raw materialand allowing the reaction to proceed while stirring.

Reaction vessels usable for the reaction are not particularly limited.The reaction vessel is preferably a reaction vessel capable of stirringso as to sufficiently mix the lignocellulosic raw material and themutant xylanase that have been added thereinto, and having a temperaturecontrol function with which the temperature can be maintained at theoptimum temperature of the mutant xylanase.

The reaction temperature may be any temperature at which the mutantxylanase can work, without particular restrictions. For example, thereaction temperature may be from 40° C. to 60° C., and preferably from40° C. to 55° C.

The pH of the solution in the saccharification reaction vessel may beany pH at which the mutant xylanase can work, without particularrestrictions. For example, the pH may be from pH 4 to pH 7, andpreferably from pH 4 to pH 6.

In the method of producing a saccharified product of lignocelluloseaccording to the invention, in addition to the mutant xylanase accordingto the invention, other enzymes may be used in combination with themutant xylanase, if necessary.

In regard to the other enzymes, enzymes, for example, cellulase,xylosidase, mannanase, pectinase, galactosidase, glucuronidase, andarabinofuranosidase, may be used in combination with the mutantxylanase. From the viewpoint of efficient production of a saccharifiedproduct of lignocellulose, cellulase is preferably used in combinationwith the mutant xylanase.

Known cellulases that decompose cellulose into glucose may be used asthe cellulase, without restrictions. Examples of the cellulase includecellulases having at least one activity selected from an endoglucanaseactivity, a cellobiohydrolase activity, or a β-glucosidase activity. Inaddition, from the viewpoint of enzymatic activity, the cellulase ispreferably an enzyme mixture having these activities.

The origin of the cellulase is not limited, and cellulases offilamentous fungi, Basidiomycetes, bacteria, and the like may be used.For example, it is possible to use one, or a mixture of two or more,selected from the group consisting of: cellulases derived from varioussources such as filamentous fungi of the genus Trichoderma, the genusAcremonium, the genus Aspergillus or the like, basidiomycetes of thegenus Irpex or the like, bacteria of the genus Aeromonas, the genusClostridium, the genus Bacillus, the genus Pseudomonas, the genusPenicillium, the genus Humicola, or the like; and cellulases produced bygenetic recombination using cellulases derived from these sources astemplates. It is also possible to directly use a cellulase formulationavailable in the general market, a cultured product of any of themicroorganisms mentioned above, or a filtrate obtained from the culturedproduct.

Among these, cellulase derived from the genus Trichoderma or cellulasederived from the genus Acremonium is preferable in consideration oftheir strong cellulose-decomposing power.

Examples of commercially available cellulases that can be used includeACCELLERASE 1000 (manufactured by Genencor Co., Ltd.), ACCELLERASE 1500(manufactured by Genencor), ACCELLERASE XC (manufactured by Genencor),ACCELLERASE XY (manufactured by Genencor), ACCELLERASE DUET(manufactured by Genencor), ACCELLERASE TRIO (manufactured by Genencor),CELLUCLAST (manufactured by Novozymes), CELLIC CTEC (manufactured byNovozymes), CELLIC HTEC (manufactured by Novozymes), CELLIC CTEC2(manufactured by Novozymes), CELLIC HTEC2 (manufactured by Novozymes),ACREMONIUM CELLULASE (manufactured by Meiji Seika Pharma Co., Ltd.),MEICELLASE (manufactured by Meiji Seika Pharma Co., Ltd.), CELLULASEAMANO A (manufactured by Amano Enzyme Co., Ltd.), CELLULASE AMANO T(manufactured by Amano Enzyme Co., Ltd.), CELLULASE DAIWA (manufacturedby Daiwa Fine Chemicals Co., Ltd.), CELLULIZER (manufactured by NagaseBiochemicals Ltd.), DRISELASE (manufactured by Kyowa Hakko Kogyo Co.,Ltd.), CELLULASE ONOZUKA (manufactured by Yakult Pharmaceutical IndustryCo., Ltd.), and CELLULOSIN (manufactured by Hankyu Bioindustry Co.,Ltd.).

The mixing ratio of the mutant xylanase to cellulase may be any mixingratio at which the production amount of reducing sugar is maximized.Preferably, the mutant xylanase is mixed in a ratio of from 20% to 60%with respect to cellulase.

The concentration of lignocellulosic raw material as a substrate to beadded into the reaction vessel and the total concentration of enzymesincluding the mutant xylanase and the other enzymes (hereinafterreferred to as simply “enzymes”) are not particularly limited.

For operations such as the transfer, charging, and the like oflignocellulosic raw material, a solid content concentration of from 8%to 30% by mass is preferable.

The enzymes to be used may be added in an amount sufficient forefficient decomposition of the substrate in view of the activity of theenzymes. The amount of the enzymes may be adjusted, as appropriate, inaccordance with, for example, the types of the enzymes.

The saccharified product produced by the method of producing asaccharified product from a lignocellulosic raw material according tothe invention and the method of producing a saccharified product from alignocellulosic raw material involving reutilization of saccharificationenzymes according to the invention may be any saccharified productderived from lignocellulose. Specific examples of the saccharifiedproduct include monosaccharides, and oligosaccharides, which consist oftwo or more sugar units. Examples of the monosaccharides includeglucose, xylose, arabinose, fructose, mannose, and galactose.

The saccharified product may be used to produce chemicals, fuels,plastics, and other products or intermediates. The saccharified productmay also be used as a raw material for fermentation for producing thesesubstances using microorganisms.

Examples of the chemicals, fuels, plastics, and other products includeethanol, isopropanol, acetone, acetate, 1,3-propanediol, butanediol,glycerol, ethylene glycol, amino acids, organic acids, furfural,polyhydroxyalkanoates, animal feeds, and xylose.

In particular, the saccharified product is highly suitable for use infermentative production of ethanol, isopropanol, lactic acid, or thelike.

[Method of Producing Mutant Xylanase for Reutilization]

The method of producing a mutant xylanase for reutilization according tothe invention includes recovering the mutant xylanase according to theinvention from a saccharification reaction solution that contains asaccharified product of lignocellulose obtained by the method ofproducing a saccharified product of lignocellulose; this process ishereinafter also referred to as a “recovery process”, and thesaccharification reaction solution mentioned above is hereinafter alsoreferred to as simply the “saccharification reaction solution”.

According to this method, the mutant xylanase according to the inventioncan be produced at low cost.

In the recovery process, the recovery method to be used may be a knownmethod. Examples thereof include a method including performingsolid-liquid separation, and recovering the enzyme using a membranedevice or other known device capable of recovering the enzyme.

Examples of methods for solid-liquid separation include centrifugationor coarse filtration of the saccharification reaction solution.

With regard to the conditions for the centrifugation or coarsefiltration, methods usually employed in the art may be used as they are.For example, in the case of centrifugation, can be performed at from500×g to 10000×g.

In the case of coarse filtration, filtration may be performed using astainless steel filter, a ceramic filter, or a resin filter membrane,each of which has an aperture size of from 0.1 μm to 2 mm.

Microfiltration using a microfiltration membrane may be performed. Inthis case, microfiltration membranes having an average pore size of from0.01 μm to 10 μm are preferably used.

Examples of methods for microfiltration using a microfiltration membraneinclude pressure filtration, vacuum filtration, cross-flow filtration,and centrifugal filtration. Among them, cross-flow filtration enablesreduction of membrane fouling.

In the case of recovering enzymes from a solution after solid-liquidseparation, examples of methods therefore include a method in which aresin column is used and a method in which a membrane device is used.

Examples of the method in which a resin column is used include knownchromatographic separation methods such as ion exchanger chromatography,reversed-phase high speed chromatography, affinity chromatography, andgel filtration chromatography.

In regard to the membrane device, recovery may be performed using, forexample, a membrane device having an ultrafiltration membrane, adialysis membrane, or the like. Among them, use of an ultrafiltrationmembrane having an average pore size of from 0.001 μm to 0.01 μm is morepreferable.

There are ultrafiltration membranes of, for example, flat membrane type,multistage flat membrane type, and hollow fiber type. Theultrafiltration described above may be any of these types. In the caseof the flat membrane type, an appropriate filtration speed can beachieved by applying a pressure to the inside of the reaction tank.Nitrogen gas, helium gas, air, or the like is preferably employed forthe application of a pressure. It is preferable to install an impellerin the reaction tank in accordance with the necessity. Stirring of theliquid using an impeller prevents fouling on the membrane surface, andenables maintenance of a more favorable filtration speed. In the casesof the multistage flat membrane type and the hollow fiber type, liquidmay be supplied from a substrate supply tank to the reaction tank usinga pump, whereby an appropriate filtration pressure and an appropriatelinear velocity are maintained, and a more favorable filtration speedcan be maintained.

Examples of filtration methods include an immersed membrane method, anultrafiltration method, and a microfiltration method. Pressurefiltration, vacuum filtration, cross-flow filtration, centrifugalfiltration, and the like are usable in both of the ultrafiltrationmethod and the microfiltration method. Filtration operations are roughlyclassified into constant pressure filtration, constant flow filtration,and filtration with a non-constant pressure and a non-constant flow;there are no particular limitations on the filtration operations in theinvention.

Examples of the material of the membrane used in the recovery process inthe invention include cellulose acetate, aromatic polyamide, polyvinylalcohol, polysulfone, polyvinylidene fluoride, polyethylene,polyacrylonitrile, ceramic, polypropylene, polycarbonate, andpolytetrafluoroethylene (TEFLON (registered trademark)). Among thesematerials, it is more preferable to use a membrane made of anacid-resistant non-cellulosic material, such as polyacrylonitrile orpolysulfone, in consideration of use of a cellulase and reaction underacidic conditions.

The saccharification reaction solution immediately after productionthereof may be used as the saccharification reaction solution for use inthe recovery process, without any pretreatment such as the solid-liquidseparation.

A mutant xylanase for reutilization produced by the method of producinga mutant xylanase for reutilization according to the invention may beused in various uses, as in the case of the mutant xylanase according tothe invention described above. The mutant xylanase for reutilization isalso usable for the method of producing a monosaccharide describedbelow.

[Method of Producing Monosaccharide]

A method of producing a monosaccharide according to the inventionincludes:

recovering the mutant xylanase according to the invention from asaccharification reaction solution containing a saccharified product oflignocellulose obtained by the method of producing a saccharifiedproduct of lignocellulose described above (the recovering of the mutantxylanase is hereinafter also referred to as the “recovery process”, andthe saccharification reaction solution mentioned above is hereinafteralso referred to as simply the “saccharification reaction solution”);and

producing a monosaccharide by contacting the recovered mutant xylanasewith a lignocellulosic raw material (hereinafter referred to as simplythe “re-saccharification process”).

In this way, the mutant xylanase according to the invention caneffectively be utilized.

To this recovery process, the recovery process described above isapplied.

In the re-saccharification process, the lignocellulosic raw material andwater are added to the enzyme solution containing the recovered mutantxylanase, and a re-saccharification reaction is performed with stirringwhile controlling the pH and the temperature. The conditions of the pHand the reaction temperature may be the same as those described in theexplanation of the method of producing a saccharified product from alignocellulosic raw material.

In the re-saccharification process, in addition to the lignocellulosicraw material and water to be additionally fed, a solid obtained by thesolid-liquid separation in the recovery process is preferably added.This makes it possible to effectively utilize unreacted lignocellulosecontained in the solid as a result of the solid-liquid separation usinga membrane device or a resin column, and the mutant xylanase that isadsorbed on the unreacted lignocellulose.

In the re-saccharification process according to the invention, eitherthe mutant xylanase or the solid product obtained as a result of thesolid-separation, or both may be additionally fed.

In the method of producing a monosaccharide according to the invention,the recovery process and the re-saccharification process may beperformed repeatedly. This makes it possible to reduce the cost forcatalyst over a period of time during which the activity of therecovered mutant xylanase is maintained.

In the method of producing a monosaccharide according to the invention,in cases in which the recovery process and the re-saccharificationprocess are repeated, the mutant xylanase according to the invention maybe newly added for the re-saccharification process. The amount of themutant xylanase to be newly added is not particularly limited, and ispreferably no more than 50% by mass of the amount of the mutant xylanaseused in the initial saccharification reaction, from the economicalviewpoint. The amount of the mutant xylanase to be newly added is morepreferably no more than 20% by mass of the amount of the mutant xylanaseused in the initial saccharification reaction from the economicalviewpoint, and the amount of the mutant xylanase to be newly added isstill more preferably no more than 10% by mass of the amount of themutant xylanase used in the initial saccharification reaction.

The monosaccharide produced by the method of producing a monosaccharideaccording to the invention may be any monosaccharide derived fromlignocellulose. Specific examples thereof include glucose, xylose,arabinose, fructose, mannose, and galactose.

[Method of Bleaching Pulp]

A method of bleaching a pulp according to the invention includescontacting the mutant xylanase with a pulp.

In the method of bleaching a pulp according to the invention, the mutantxylanase described above, which stably works over a long period of timeeven under a condition in which enzymes easily inactivate, is used, and,therefore, the bleaching can be performed under a condition in whichenzymes generally easily inactivate, and pulp can be bleached with highefficiency.

The pulp used in the process of contacting the mutant xylanase with apulp may be a wood pulp or a non-wood pulp. Examples of the wood pulpinclude those made from softwood or hardwood raw materials. Examples ofthe non-wood pulp include those made from raw materials such as bagasse,which is a cane trash of sugar cane left after squeezing, straw, hemp,and cotton. Further examples of the non-wood pulp include waste paperpulp made from waste paper, such as newspaper or magazine.

These pulps are roughly classified into mechanical pulps obtained byextracting fibers from a raw material using a physical force andchemical pulps obtained by extracting fibers by chemical treatment.Examples of mechanical pulps include ground pulp, refiner ground pulp,thermomechanical pulp, and chemi-thermo-mechanical pulp. Examples ofchemical pulps include kraft pulp, alkaline pulp, and sulfite pulp.

In the process of contacting the mutant xylanase with a pulp, inaddition to the mutant xylanase, another hemicellulase or ligninase mayadditionally be used. This heightens the degree of pulp bleaching.

The origins of the hemicellulase and the ligninase are not particularlylimited, and examples of the origins include filamentous fungi,basidiomycetes, and bacteria.

The method of bleaching a pulp according to the invention preferablyfurther includes a delignification treatment process and a bleachingprocess, in addition to the process of contacting the mutant xylanasewith a pulp.

In the present specification, the delignification treatment process maybe any method that aims to positively remove lignin from a pulp, andmethods that have been practiced from the past may be used. Examplesthereof include a method described in JP-A No. 2004-263310.

In the present specification, the bleaching process may be any processperforms bleaching treatment on the pulp, and the scope thereofgenerally encompasses process aiming at, for example, removal of ligninremaining in the pulp or improvement in the whiteness of the pulp. Thebleaching process is a process that follows the delignificationtreatment process, and methods that have been practiced from the pastmay be used. Examples thereof include a method described in JP-A No.2010-1594.

In cases in which the process of contacting the mutant xylanaseaccording to the invention with a pulp is performed in combination withthe delignification treatment process and the bleaching process, theprocess of contacting the mutant xylanase with a pulp may be performedat any point in time during processes from the delignification treatmentprocess to the bleaching process. Specifically, the process ofcontacting the mutant xylanase with a pulp may be performed before orafter the delignification treatment process, or before or after thebleaching process. Alternatively, the process of contacting the mutantxylanase with a pulp may be performed simultaneously with thedelignification treatment process or the bleaching process.

Preferably, the process of contacting the mutant xylanase according tothe invention with a pulp is performed as a part of the bleachingprocess. In particular, from the viewpoint of enabling the ability ofthe mutant xylanase according to the invention to be maximally exerted,the process of contacting the mutant xylanase with a pulp is morepreferably performed at a stage of the bleaching process at which thelignin content is small.

Alternatively, the process of contacting the mutant xylanase with a pulpmay also be used, for example, as a part of the bleaching process inwhich, from among bleaching processes described above, chemicalbleaching is performed using chlorine, chlorine dioxide, nitrogendioxide, a hypochlorite, oxygen, hydrogen peroxide, ozone, or the like.

[Detergent]

A detergent according to the invention includes the mutant xylanasedescribed above, and may further include other components, as necessary.

The detergent according to the invention has improved performance due toinclusion of the mutant xylanase, which exhibits stable activity evenunder severe conditions in which enzymes easily inactivate.

The scope of the detergent according to the invention encompassesvarious detergents such as laundry detergents and detergents forautomatic dishwashers. The detergent according to the invention may beused as detergents for home use and industrial use. The detergentaccording to the invention is also usable as a modifier for fiberproducts for clothing.

When the detergent according to the invention is used as a modifier fora fiber product for clothing, the fiber product for clothing to whichthe detergent is applied may be, for example, cotton fibers, hempfibers, or cellulose-containing fibers such as rayon or tencel.

The detergent according to the invention may further include otherenzymes in addition to the mutant xylanase, in accordance with the uses.Enzymes known in the art may be used as the other enzymes. Examplesthereof include protease, cellulase, amylase, and lipase. The origins ofthe other enzymes are not limited, and examples thereof includefilamentous fungi, basidiomycetes, and bacteria.

The detergent according to the invention may also include components,other than the other enzymes mentioned above, that are usually used indetergents, examples of which include surfactants, cleaning aids,bleaching agents, and fluorescent agents.

Examples of the surfactant include anionic surfactants, nonionicsurfactants, amphoteric surfactants, and cationic surfactants. Anionicsurfactants and nonionic surfactants are preferable.

Examples of the anionic surfactants include sodium salts of fatty acids(soap), sodium α-sulfonated fatty acid ester, sodium linear alkylbenzene sulfonate (LAS), sodium alkyl sulfate (AS), sodium alkyl ethersulfate (AES), sodium α-olefin sulfonate (AOS), and sodium alkylsulfonate.

Examples of the nonionic surfactants include polyoxyalkylene alkyl ether(AE), polyoxyethylene alkyl phenyl ether (APE), sucrose fatty acid saltesters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acidesters, polyoxyethylene fatty acid esters, and alkanol amides.

Examples of the cleaning aids include alkali buffers, divalent metal ionscavengers, and anti-redeposition agents. Specific examples thereofinclude polyphosphates such as tripolyphosphate and pyrophosphate,aluminosilicates such as A-type zeolite, carbonates such as sodiumcarbonate, sodium sesquicarbonate, and sodium hydrogen carbonate,polymers such as polyethylene glycol, carboxylic acid-based polymers,polyvinyl alcohol, polyvinyl pyrrolidone, and polyglycidyl acid salts,cellulose derivatives such as carboxymethyl cellulose, andaminocarboxylic acid-based polymers such as polyaspartic acid.

Examples of the bleaching agents include sodium hypochlorite,dichloroisocyanurates, sodium chlorite, hydrogen peroxide, sodiumpercarbonate, sodium perborate, peracetic acid, hydrosulfite, andthiouric acid dioxide.

Examples of the fluorescent agents include bis-(triazinylamino)stilbenedisulfonic acid derivatives and bis-styryl biphenyl derivatives.

The detergent according to the invention may be combined with thesurfactant, the cleaning aid, the bleaching agent, the fluorescentagent, or the like, and produced according to ordinary methods.

The form of the detergent may be selected in accordance with the uses,and may be, for example, liquid, powder, granules, paste, or solid.

[Animal Feed]

An animal feed according to the invention includes the mutant xylanasedescribed above, and may further include other components, as necessary.

The animal feed according to the invention includes the mutant xylanasethat exhibits stable activity even under severe conditions in whichenzymes easily inactivate. As a result, the absorption efficiency ofplant nutrients in animals that have eaten the animal feed according tothe invention is improved, and the digestibility thereof in the stomachof the animals is also improved, due to the decomposition of plantfibers abundant in the animal feed.

The content of the mutant xylanase in the animal feed according to theinvention should be an amount capable of improving the digestibility ofthe animal feed in the stomach of animals, but the content is notparticularly limited in other respects.

Examples of the animal feed include xylan-containing ready-made animalfeeds, and grains. Among the grains, wheat, corn, rye, barley, oats,triticale, rice, and sorghum are particularly preferable.

The mutant xylanase in the animal feed according to the invention may beused in combination with other feed additives and/or other enzymes.

Examples of other feed additives include vitamin feed additives, mineralfeed additives, amino acid feed additives, and permeable protectiveagents.

Examples of other enzymes include cellulase, amylase, and protease. Theorigins of these enzymes are not limited, and enzymes derived fromfilamentous fungi, basidiomycetes, bacteria, or the like may be used.

The animal feed according to the invention is usable for a wide range ofanimals. Preferable examples of the animals include poultry such aschickens, turkeys, ducks, and geese, ruminants such as cows, horses, andsheep, boars and pigs such as pigs, rodents such as rabbits, and fishes.

The animal feed according to the invention may be produced by any methodas long as the animal feed includes the mutant xylanase, and the methodfor producing the animal feed is not particularly limited. Addition ofthe mutant xylanase into animal feed may be performed at any stageselected from before the production of the animal feed, during theproduction of the animal feed, or at the final stage of the productionof the animal feed. The mutant xylanase may be directly added to aready-made animal feed that has been formed into a pellet form or a mashform. Alternatively, the mutant xylanase may be incorporated into ananimal feed by being directly added into drinking water.

[Bread-Making Modifier]

A bread-making modifier according to the invention includes the mutantxylanase described above, and may further include other components, asnecessary.

The bread-making modifier according to the invention includes the mutantxylanase described above, which exhibits stable activity even undersevere conditions in which enzymes easily inactivate. Due to theinclusion of the mutant xylanase, the bread-making modifier according tothe invention exhibits stable activity even during the fermentationprocess in bread making, which is carried out at from 35° C. to 40° C.for from 1 hour to 2 hours, whereby hemicellulose contained in the flourcan be decomposed, and the quality of bread making can be modified.

The bread-making modifier according to the invention may include otherbread-making modifiers, in addition to the mutant xylanase. Examples ofthe other bread-making modifiers include monoglycerides, organic acidmonoglycerides, glycerin fatty acid esters, propylene glycol fatty acidesters, sorbitan fatty acid esters, phospholipids, ascorbic acids andderivatives thereof, organic acids, amino acids, and salts.

In regard to the type of bread to which the bread-making modifieraccording to the invention is to be added, the bread may be any breadthat is produced by mixing ingredients for the bread and furtherperforming kneading, fermentation, baking, and the like. Examplesthereof include, besides white bread, special bread, stuffed bread,sweet bun, steamed bread, pancakes, and doughnuts.

Examples of ingredients for these breads include flour, water contentssuch as water and dairy products, yeast, sugars, common salt, and oilsand fats (such as shortening, lard, margarine, butter, and liquid oil).If necessary, eggs, seasonings (such as glutamic acids and nucleicacids), baking powder, flavors, or the like may also be added. In casesin which flour is a main raw material, rye flour, rice flour, or thelike may also be used in combination with the flour. In the presentspecification, the term “dough” means a material obtained by mixing andkneading the bread ingredients mentioned above.

Methods for producing bread may be commonly-employed methods thatinclude a fermentation process, without particular limitations. Forexample, a straight dough method, a sponge and dough method, and apre-ferment and dough method may be used.

For the fermentation process, commonly-employed methods may be used. Thefermentation process is preferably performed at from 35° C. to 40° C.for from 1 hour to 2 hours since the fermentation time can be shortenedby setting the fermentation temperature relatively high as compared toroom temperature.

The bread-making modifier according to the invention may be, forexample, mixed as powder with a raw material such as flour, or dissolvedin water before use, or added as powder or liquid at a certain stage inthe process.

Although embodiments of the invention are described above, theseembodiments are merely examples of the invention, and variousconfigurations, other than those described above, may also be employed.

EXAMPLES

The invention will be described in more detail with reference to thefollowing Examples. However, the invention is by no means limited to theexamples below. The percentages indicating the amounts of componentsincluded in the compositions in the examples are percentages by mass,unless otherwise specified.

Example 1 Method for Measuring Xylanase Activity (Standard Assay)

The amount of reducing sugars released by the hydrolysis of xylan wasmeasured by the DNS method (Bailey et al., 1992), to determine xylanaseactivity.

The substrate used for evaluation was a supernatant prepared byvigorously mixing together a 100 mM sodium citrate buffer solution (pH4.5) with 1% (w/w) birchwood xylan (manufactured by Sigma-AldrichCorporation) and then centrifuging at 5,000×g for 15 minutes.

Xylanase was mixed with this substrate solution such that the amount ofxylanase was 0.1% (w/w) of that of the substrate solution, and areaction was allowed to proceed with stirring at 45° C. for 30 minutes.The amount of reducing sugars in the resulting reaction solution wasmeasured, to determine the xylanase activity.

Example 2 Production of Xylanase Mutant by Site-Directed Mutagenesis andEvaluation Thereof

(1) Construction of Expression Vectors: YEp-GAPDHp-GAs-TVX andYEp-GAPDHp-GAs-ACX

(a) Obtainment of Promoter Sequence

Using a genomic DNA sequence of Saccharomyces cerevisiae as a template,a promoter sequence (GenBank Accession Number: A35397.1) ofglyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as“GAPDH”) was obtained by PCR. The primer sequences used in the PCR arepresented as SEQ ID Nos. 55 and 56 in Table 4 below.

(b) Obtainment of Signal Sequence

Using a genomic DNA sequence of Rhizopus oryzae as a template, a signalsequence of a glucoamylase gene (GenBank Accession Number: D00049.1) wasobtained by PCR. The primer sequences used in the PCR are presented asSEQ ID NOs. 57 and 58 in Table 4 below.

(c) Ligation of Promoter Sequence and Signal Sequence

The DNA sequences amplified by the PCR were purified using aphenol/chloroform solution, and recovered through ethanol precipitation.The purified promoter sequence and signal sequence were digested with arestriction enzyme BglII, and thereafter individually subjected toagarose electrophoresis, and fragments that included desired DNAs wereseparated and purified. The fragments thus obtained were ligated using aDNA ligase (manufactured by Takara Shuzo Co., Ltd). The ligated productis hereinafter abbreviated to “GAPDHp-GAs”.

(d) Amplification of T. Viride-Derived Xylanase II Gene

Using a genomic DNA sequence of T. viride as a template, the full lengthof a T. viride-derived xylanase II gene (a base sequence encoding thexylanase gene as well as a secretory signal sequence) was obtained byPCR. The primer sequences used in the PCR are presented as SEQ ID NO: 59and SEQ ID NO 60 in Table 4 below. The obtained sequence is presented asSEQ ID NO: 74 in the Sequence Listing.

(e) Amplification of A. Cellulolyticus-Derived Xylanase Gene

Using a genomic DNA sequence of A. cellulolyticus as a template, thefull length of an A. cellulolyticus-derived xylanase gene (a basesequence encoding the xylanase gene as well as a secretory signalsequence) was obtained by PCR. The primer sequences used in the PCR arepresented as SEQ ID NOs: 61 and 62 in Table 4 below. The obtainedsequence is presented as SEQ ID NO: 75 in the Sequence Listing.

(f) Ligation of Promoter Sequence, Signal Sequence, and Xylanase Gene

Using the T. viride-derived xylanase II obtained in (d) as a template,the base sequence encoding the xylanase gene, excluding the signalsequence, was obtained by PCR. The primers used in the PCR are presentedas SEQ ID NO: 63 and SEQ ID NO 64 in Table 4 below. Thereafter, theobtained fragment was purified and digested with a restriction enzymeSacI, and then ligated to GAPDHp-GAs fragment. The ligation product ishereinafter abbreviated to “GAPDHp-GAs-TVX”.

Similar to the above, also with respect to the A. cellulolyticus-derivedxylanase I obtained in (e), the gene at the secretory protein portionthereof was obtained by PCR, and, after purification, digested with arestriction enzyme SacI and ligated to GAPDHp-GAs fragment. The ligationproduct is hereinafter abbreviated to “GAPDHp-GAs-ACX”. The primers usedin the PCR are presented as SEQ ID NO: 65 and SEQ ID NO 66 in Table 4below.

Here, the methods for the purification and ligation of DNA fragments inthis step are the same as those in the step (c).

(g) Introduction into Expression Vector

The GAPDHp-GAs-TVX fragment and a multicopy expression vector YEp24(ATCC 7769) for budding yeast were digested with restriction enzymesXmaI and BamHI (the former producing a fragment of about 1.3 kbp and thelatter producing a fragment of about 7.4 kbp), and, after purification,ligated to each other to obtain a plasmid for producing a T.viride-derived xylanase II mutant (hereinafter abbreviated toYEp-GAPDHp-GAs-TVX). Similar to the above, the fragment GAPDHp-GAs-ACXand YEp24 were digested with the restriction enzymes XmaI and BamHI (theformer producing a fragment of about 1.5 kbp and the latter producing afragment of about 7.4 kbp), and, after purification, ligated to eachother to obtain an expression vector for producing an A.cellulolyticus-derived xylanase I mutant (hereinafter abbreviated toYEp-GAPDHp-GAs-ACX).

The methods for the purification and ligation of the DNA fragments inthis step are the same as those in the step (c). The YEp24 is availablefrom the American Type Culture Collection, which is a bank of cells andmicroorganisms.

TABLE 4 SEQ ID NO: 55 GACTAGCCCGGGTCGAGTTTATCATTATC SEQ ID NO: 56GACGAGAGATCTCCATTTTGTTTATTTATGTG SEQ ID NO: 57GACTAGAGATCTATGCAACTGTTCAATTTGCC SEQ ID NO: 58CAGCATGAGCTCAGCAGAAACCAGCAAAG SEQ ID NO: 59ATGGTTTCCTTCACCTCCCTCCTCGCCGGC SEQ ID NO: 60TTAGCTGACGGTAATAGAAGCAGAGCCAGA SEQ ID NO: 61ATGGGCATCTCATCTATTCTTCTCTCTGCT SEQ ID NO: 62CTATTGGCACTGACTGTAGTAAGCGTTAAA SEQ ID NO: 63GATTAGGAGCTCCAGACGATTGGTCCCG SEQ ID NO: 64 GACTAGGGATCCTTAGCTGACGGTAATAGSEQ ID NO: 65 GATTATGAGCTCGCTGAGGCGATCAACTAC SEQ ID NO: 66GATTAGGGATCCCTATTGGCACTGACTGTAG

(2) Site-Directed Mutagenesis

Mutants used in examples of the invention had mutations introduced usinga LA PCR in vitro Mutagenesis Kit manufactured by Takara Shuzo Co., Ltd.and using the expression vectors constructed in step (1) as templates

The primers used were synthesized oligonucleotides.

PCR was performed using the expression vector YEp-GAPDHp-GAs-TVX as atemplate and using SEQ ID NO: 5 and SEQ ID NO 6, SEQ ID NO: 7 and SEQ IDNO 8, SEQ ID NO: 9 and SEQ ID NO 10, and SEQ ID NO: 11 and SEQ ID NO 12,which are given in Table 5 below, as primers, to obtain a mutantxylanase expression vector YEp-GAPDHp-GAs-TVX01.

In addition, using the expression vector YEp-GAPDHp-GAs-ACX as atemplate and using the sequences of SEQ ID NO: 21 and SEQ ID NO 22,which are given in Table 5 below, as primers, YEp-GAPDHp-GAs-L154M wasobtained which had a substitute amino acid residue that was a methioninesubstituted for a leucine residue at position 154 of SEQ ID NO: 2 in theSequence Listing.

Similar to the above, YEp-GAPDHp-GAs-ACX01 was obtained using theexpression vector YEp-GAPDHp-GAs-ACX as a template and using thesequences of SEQ ID NO: 13 and SEQ ID NO 14, the sequences of SEQ ID NO:15 and SEQ ID NO 16, the sequences of SEQ ID NO: 17 and SEQ ID NO 18,the sequences of SEQ ID NO: 19 and SEQ ID NO 20, the sequences of SEQ IDNO: 21 and SEQ ID NO 22, the sequences of SEQ ID NO: 23 and SEQ ID NO24, the sequences of SEQ ID NO: 25 and SEQ ID NO 26, the sequences ofSEQ ID NO: 27 and SEQ ID NO 28, and the sequences of SEQ ID NO: 29 andSEQ ID NO 30, which are given in Table 5 below, as primers.

Similar to the above, YEp-GAPDHp-GAs-ACX02 was obtained using theexpression vector YEp-GAPDHp-GAs-ACX as a template and using thesequences of SEQ ID NO: 15 and SEQ ID NO 16, the sequences of SEQ ID NO:21 and SEQ ID NO 22, the sequences of SEQ ID NO: 31 and SEQ ID NO 32,and the sequences of SEQ ID NO: 33 and SEQ ID NO 34, which are given inTable 5 below, as primers.

Similar to the above, YEp-GAPDHp-GAs-ACX03 was obtained using theexpression vector YEp-GAPDHp-GAs-ACX as a template and using thesequences of SEQ ID NO: 21 and SEQ ID NO 22, the sequences of SEQ ID NO:31 and SEQ ID NO 32, the sequences of SEQ ID NO: 35 and SEQ ID NO 36,the sequences of SEQ ID NO: 37 and SEQ ID NO 38, and the sequences ofSEQ ID NO: 39 and SEQ ID NO 40, which are given in Table 5 below, asprimers.

Competent cells of Escherichia coli HB101 (manufactured by Toyobo Co.,Ltd.) were transformed with the plasmids that contained mutantxylanases, to obtain transformants.

Plasmids were prepared from the bacterial cells using an alkaline-SDSextraction method, and the base sequences of the xylanase gene portionsthereof were determined using a DNA sequencer, as a result of which theintroduction of amino acid substitutions in the xylanase gene-encodingregion of each of YEp-GAPDHp-GAs-TVX and YEp-GAPDHp-GAs-ACX, which aretemplates, was confirmed.

TABLE 5 SEQ ID NO: 5 GTACACCCTCGGCCCCGGCGGCCAG SEQ ID NO: 6CGGGGCCGAGGGTGTACGTCACGCC SEQ ID NO: 7 CAAGAACAGGGTCATCAACTTCTCGSEQ ID NO: 8 GATGACCCTGTTCTTGGTGCCGG SEQ ID NO: 9CGTGACGTTCACCCTCGGCCCCGGC SEQ ID NO: 10 CGAGGGTGAACGTCACGCCGCCGTGSEQ ID NO: 11 CTCGGGCAGCTTTGTCGGCGGCAAG SEQ ID NO: 12CGACAAAGCTGCCCGAGTTGGACCAG SEQ ID NO: 13 CATCAACTACGATACGCAGGGGGACSEQ ID NO: 14 CCCTGCGTATCGTAGTTGATGGAG SEQ ID NO: 15GATACGCAGAGGGACTTTGTGGTGG SEQ ID NO: 16 CAAAGTCCCTCTGCGTATCGTAGTTGSEQ ID NO: 17 GATACCAGTCTGTCGGCACACACAAG SEQ ID NO: 18GCCGACAGACTGGTATCCGTCGTG SEQ ID NO: 19 GATCCGCCGAAGCCCCCGGACGAGSEQ ID NO: 20 GGGGGCTTCGGCGGATCGAGATGTAC SEQ ID NO: 21CAGGCGGGCATGAATCTCGGCACAATG SEQ ID NO: 22 CCGAGATTCATGCCCGCCTGCGCCCSEQ ID NO: 23 GCAGCGGCACTGGACAAATCTCGCTC SEQ ID NO: 24GATTTGTCCAGTGCCGCTGCCGCTCC SEQ ID NO: 25 CACGGGTCACACCAGCACGAGCACSEQ ID NO: 26 GTGCTGGTGTGACCCGTGGGTGTG SEQ ID NO: 27GTCACACCAACACGAGCACCGCTCC SEQ ID NO: 28 GTGCTCGTGTTGGTGTGACCCGTGGSEQ ID NO: 29 CAATGCGGAGAAATTGGCTGGACCGG SEQ ID NO: 30CCAGCCAATTTCTCCGCATTGTCCCC SEQ ID NO: 31 CTTTCTCCGTCAACTACAATACGCSEQ ID NO: 32 GTAGTTGACGGAGAAAGAACCCG SEQ ID NO: 33GTCAACTACGATACGCAGGGGGACTTTG SEQ ID NO: 34 CCCTGCGTATCGTAGTTGACGGAGAAAGSEQ ID NO: 35 CTCCTTCACGGCCTCGGGTCGGGTG SEQ ID NO: 36CCGAGGCCGTGAAGGAGCCGCTGTAG SEQ ID NO: 37 CGCTTACCACAGTCAGTGCCAATAGSEQ ID NO: 38 CACTGACTGTGGTAAGCGTTAAAGTAC SEQ ID NO: 39CAGTCAGAGCCAATAGGGATCCTC SEQ ID NO: 40 CCTATTGGCTCTGACTGTAGTAAGC

(3) Transformation into Yeast and Production of Mutant Xylanase

Competent cells of Saccharomyces cerevisiae BY4741 were transformed withthe mutant xylanase-containing expression vectors constructed in step(2) using a FAST-YEAST TRANSFORMATION KIT (G-Biosciences), and culturedon an SD-Ura agar medium (0.67% Yeast-nitrogen base without amino acids(Difco Co., Ltd.), 2% glucose, 0.5% casamino acid, 0.077%-Ura DOSupplement (Clontech Co., Ltd.), 2% agar, and deionized water) at 30° C.for 48 hours. The resultant mutants are listed in Table 6.

TABLE 6 Name of SEQ ID Amino acid Before After mutant NO: No. mutationmutation TVX01 1 27 Tyr Phe 1 29 Asn Leu 1 44 Asn Ser 1 58 Lys Arg ACX012 33 Asn Asp 2 36 Gly Arg 2 90 Thr Ser 2 132 Gln Arg 2 154 Leu Met 2 174Ser Thr 2 195 Pro His 2 197 Ser Asn 2 217 Gly Glu ACX02 2 30 Ile Val 233 Asn Asp 2 36 Gly Arg 2 154 Leu Met ACX03 2 30 Ile Val 2 59 Ser Thr 2154 Leu Met 2 239 Tyr His 2 242 Cys Ser

(4) Evaluation of Stability of Mutant Xylanases

The obtained colony was inoculated into an SD-Ura liquid medium (themedium composition being the same as the above-mentioned mediumcomposition except that glucose content was 4%, that the agar was notcontained, and that the SD-Ura medium was a liquid medium), and wassubjected to pre-cultivation at 30° C. for 24 hours. Thereafter, theresultant pre-culture was inoculated in an amount of 2%, and subjectedto main cultivation for 48 hours. Then, the supernatant, which containedthe mutant xylanase, was centrifuged, and then subjected to heatingtreatment at from 50° C. to 55° C., and residual activity of the enzymewas measured according to the measurement method described in Example 1.

<TVX01>

The mutant xylanase produced by the method described in step (2) inExample 2 was subjected to heat treatment at 50° C. for a length of timevaried from 0 hour to 72 hours, and then measured with respect to theresidual activity thereof using the measurement method described inExample 1. The results are given in Table 7. The symbol WT representswild type, and the symbols F, L, S, and R respectively represent, in theamino acid sequence of SEQ ID NO: 1 in the Sequence Listing,substitution of a tyrosine residue at position 27 with phenylalanine,substitution of an asparagine residue at position 29 with a leucineresidue, substitution of an asparagine residue at position 44 with aserine residue, and substitution of a lysine residue at position 58 withan arginine residue. For the production of a mutant xylanase (Table 7(e)) in which a tyrosine residue at position 27 was substituted with aphenylalanine residue, primers of SEQ ID NO: 41 and SEQ ID NO 42 givenin Table 8 were used.

TABLE 7 Initial Rate of Reaction Name of Before Heat Treatment ResidualActivity [%] No. Xylanase (Relative to WT) 0 hours 24 hours 48 hours 72hours (a) WT 1.00 100% 0% 0% 0% (b) FLSR (TVX01) 0.70 100% 97% 86% 75%(c) FLR 0.48 100% 84% 74% 55% (d) LR 0.66 100% 0% 0% 0% (e) F 0.79 100%0% 0% 0% (f) L 0.57 100% 0% 0% 0% (g) S 0.99 100% 0% 0% 0% (h) R 0.96100% 0% 0% 0%

TABLE 8 SEQ ID NO: 41 CGTGACGTTCACCAATGGCCCCGGC SEQ ID NO: 42CATTGGTGAACGTCACGCCGCCGTG

The mutant xylanases in which a specific amino acid residue or specificamino acid residues were replaced by substitute amino acid residues thatare believed to provide stabilization against heat and described in WO2007/115391 pamphlet, WO 2001/27252 pamphlet, and WO 2005/108565pamphlet completely inactivated after 24 hours, as demonstrated in rows(d), (f), and (h) in Table 7.

In addition, as shown in rows (e) and (g) in Table 7, the mutantxylanase having a substitute amino acid residue that is a phenylalanineresidue substituted for a tyrosine residue at position 27 or the mutantxylanase having a substitute amino acid residue that is a serine residuesubstituted for an asparagine residue at position 44 also completelyinactivated after 24 hours.

However, in the case of the mutant xylanase having substitute amino acidresidues that are a leucine residue substituted for an asparagineresidue at position 29 and an arginine residue substituted for a lysineresidue at position 58 (row (d) in Table 7), further substitution of atyrosine residue at position 27 with a phenylalanine residue resulted inimprovement of residual activity by and provision of a residual activityof 50% or higher even after 72 hours (row (c) in Table 7).

Furthermore, substitution of an asparagine residue at position 44 inthis mutant xylanase with a serine residue resulted in provision of anactivity close to that of the wild type (row (a) in Table 7) and aresidual activity of 70% even after 72 hours (row (b) in Table 7).

Many of the mutants that include the four mutation sites according tothe invention had properties nearly equivalent to those of TVX01.Specific examples of those mutants include mutants that includes thefour mutation sites as well as includes, in the amino acid sequence ofSEQ ID NO: 1 in the Sequence Listing, substitution of a glycine residueat position 47 with a cysteine residue, substitution of a glutamineresidue at position 52 with a lysine residue, substitution of a valineresidue at position 59 with an isoleucine residue, substitution of anasparagine residue at position 67 with an aspartic acid residue,substitution of an asparagine residue at position 69 with an isoleucineresidue, substitution of a serine residue at position 80 with an alanineresidue, substitution of an asparagine residue at position 97 with anaspartic acid residue, substitution of a leucine residue at position 105with a methionine residue, substitution of a threonine residue atposition 109 with an alanine residue, substitution of a threonineresidue at position 120 with an arginine residue, substitution of athreonine residue at position 143 with an isoleucine residue,substitution of an asparagine residue at position 151 with a serineresidue, substitution of a serine residue at position 161 with a leucineresidue, or substitution of a serine residue at position 186 with athreonine residue.

<L154M>

Using the method described in step (3) in Example 2, yeast wastransformed with the expression vector that includes a nucleic acidrepresented by the base sequence encoding the amino acid sequence of amutant xylanase, and that was produced by the method described in step(2) in Example 2. The yeast that produces the mutant xylanase wassubjected to cultivation in liquid. The supernatant of the culturesolution was subjected to heat treatment at 50° C. for 24 hours, andthen residual activity was measured by the measurement method describedin Example 1.

The residual activity of the mutant xylanase was 50%. The mutantxylanase also exhibited an initial rate of reaction before heattreatment that is 1.13 times that of the wild type.

<ACX01, ACX02, and ACX03>

Using the method described in step (3) in Example 2, yeast wastransformed with the expression vector that includes a nucleic acidrepresented by the base sequence encoding the amino acid sequence of amutant xylanase, and that was produced by the method described in step(2) in Example 2. The yeast that produces the mutant xylanase wassubjected to cultivation in liquid. The supernatant of the culturesolution was subjected to heat treatment at 50° C. for a length of timevaried from 0 to 48 hours, and then residual activity was measured bythe measurement method described in Example 1. The results are given inTable 9. In Table 9, WT represents wild type.

The liquid medium used in this process is an SD medium (without Ura)that contained 4% of glucose.

TABLE 9 Initial Rate of Reaction Before Heat Treatment Residual Activity(%) No. Name of Xylanase (Relative to WT) 0 hours 16 hours 24 hours 48hours (i) WT 1.00 100% 0% 0% 0% (j) ACX01 2.27 100% 89% 76% 59% (k)ACX02 0.92 100% 99% 92% 66% (l) ACX03 0.80 100% 85% 79% 61%

The wild-type A cellulolyticus-derived xylanase completely inactivatedafter heat treatment for 16 hours (row (i) in Table 9). In contrast, themutant xylanases exhibited an improved residual activity, and the mutantxylanases exhibited a residual activity of 50% or higher even after 48hours (rows (j), (k), or (l) in Table 9). ACX02 and ACX03 (rows (k) and(l) in Table 9) also exhibited initial rates of reaction before heattreatment that are nearly equivalent to that of the wild-type xylanase,and ACX01 exhibited an activity nearly twice as high as that of thewild-type xylanase (row (j) in Table 9).

Many of the mutants that have all of the mutation sites contained inACX01 exhibit properties nearly equivalent to those of ACX01. Specificexamples of the mutants include a mutant that includes the mutationsites contained in ACX01, and further includes, in the amino acidsequence of SEQ ID NO:3 in the Sequence Listing, substitution of aserine residue at position 133 with an asparagine residue andsubstitution of a glutamine residue at position 176 with an arginineresidue.

Similar to the above, in the case of ACX02, many of the mutants havingall of the mutation sites contained in ACX02 exhibit properties nearlyequivalent to those of ACX02. Specific examples of the mutants include amutant that includes the mutation sites contained in ACX02, and furtherincludes, in the amino acid sequence of SEQ ID NO: 3 in the SequenceListing, substitution of a threonine residue at position 90 with aserine residue, substitution of a glutamine residue at position 132 withan arginine residue, substitution of a serine residue at position 133with an asparagine residue, substitution of a serine residue at position174 with a threonine residue, substitution of a proline residue atposition 195 with a histidine residue, substitution of a glutamineresidue at position 176 with an arginine residue, substitution of aserine residue at position 197 with asparagine residue, and substitutionof a glycine residue at position 217 with a glutamic acid residue.

Further, many of the mutants that include all of the mutation sitescontained in ACX03 exhibit properties nearly equivalent to those ofACX03. Specific examples of the mutants include a mutant that includesall the mutation sites contained in ACX03, and further includessubstitution of a glutamine residue at position 176 in the amino acidsequence of SEQ ID NO: 3 in the Sequence Listing with an arginineresidue.

Comparative Examples

It is known that introduction of mutations for improving the heatresistance of an enzyme usually largely decreases the initial rate ofreaction, or completely inactivates the enzyme. Also in thisapplication, it was observed that obtained mutants exhibited a decreasedinitial rate of reaction in most cases although the heat resistancethereof was improved. One example thereof is given in Table 10, in whichWT represents wild type and MT represents a mutant.

These mutants were produced using the technique described in step (2) inExample 2.

MT1 was obtained using the plasmid YEp-GAPDHp-GAs-TVX as a template andusing the sequences of SEQ ID NO: 43 and SEQ ID NO 44, the sequences ofSEQ ID NO: 45 and SEQ ID NO 46, and the sequences of SEQ ID NO: 47 andSEQ ID NO 48, which are given in Table 11 below, as primers.

MT2 was obtained using the plasmid YEp-GAPDHp-GAs-TVX as a template andusing the sequences of SEQ ID NO: 7 and SEQ ID NO 8, the sequences ofSEQ ID NO: 47 and SEQ ID NO 48, and the sequences of SEQ ID NO: 49 andSEQ ID NO 50, which are given in Table 11 below, as primers.

MT3 was obtained using the plasmid YEp-GAPDHp-GAs-ACX as a template andusing the sequences of SEQ ID NO: 51 and SEQ ID NO 52 and the sequencesof SEQ ID NO: 53 and SEQ ID NO 54, which are given in Table 11 below, asprimers.

The mutation sites of MT1 in Table 10 indicate that, in the amino acidsequence of SEQ ID NO: 1 in the Sequence Listing, a phenylalanineresidue is substituted for a tyrosine residue at position 13 (Tyr13Phe),a cysteine residue is substituted for a glycine residue at position 47(Gly47Cys), and a serine residue is substituted for an asparagineresidue at position 151 (Asn151Ser).

The mutation sites of MT2 indicate that, in the amino acid sequence ofSEQ ID NO: 1 in the Sequence Listing, an isoleucine residue issubstituted for a valine residue at position 46 (Val46Ile), an arginineresidue is substituted for a lysine residue at position 58 (Lys58Arg),and a serine residue is substituted for an asparagine residue atposition 151 (Asn151Ser).

The mutation sites of MT3 indicate that, in the amino acid sequence ofSEQ ID NO: 2 in the Sequence Listing, a cysteine residue is substitutedfor a serine residue at position 100 (Ser100Cys), and a cysteine residueis substituted for an asparagine residue at position 144 (Asn144Cys).

TABLE 10 Initial Rate of Residual Activity Reaction Before (Relative toHeat Treatment That Before (Relative to WT) Name of Mutation HeatTreatment) Before Xylanase Sites After 16 hours Heat Treatment WT (T.viride) —  5% 1.00 MT1 (T. viride) Tyr13Phe + 69% 0.38 Gly47Cys +Asn151Ser MT2 (T. viride) Val46Ile + 39% 0.33 Lys58Arg + Asn151Ser WT(A. — 63% 1.00 Cellulolyticus) MT3 (A. Ser100Cys + 84% 0.25Cellulolyticus) Asn144Cys

TABLE 11 SEQ ID NO: 43 CAACAACGGCTTCTTCTACTCGTACTG SEQ ID NO: 44CGAGTAGAAGAAGCCGTTGTTGAAGCC SEQ ID NO: 45 CAACTTTGTCTGCGGCAAGGGATGGSEQ ID NO: 46 CCATCCCTTGCCGCAGACAAAGTTG SEQ ID NO: 47CTCCGTCAGCACGGCGAACCAC SEQ ID NO: 48 GTGGTTCGCCGTGCTGACGGAGSEQ ID NO: 49 GCAACTTTATCGGCGGCAAGGGATG SEQ ID NO: 50CTTGCCGCCGATAAAGTTGCCCGAG SEQ ID NO: 51 CACTGTGACGTGCGACGGCGGCACSEQ ID NO: 52 CCGCCGTCGCACGTCACAGTGCC SEQ ID NO: 53CCGTGCAGTGCCACTTCAATGCC SEQ ID NO: 54 CATTGAAGTGGCACTGCACGGTAAC

Example 3 Mass Production of TVX01 and ACX02 Using T. viride as Host

(1) Mass Production of TVX01 Using T. viride

(a) Construction of Plasmid TVX01-pCB1

Using the base sequence encoding the mutant xylanase TVX01 obtained inExample 2 as a template, the DNA sequence of the xylanase portion wasobtained by PCR.

The primers used in the PCR were presented as SEQ ID NO: 67 and SEQ IDNO 68 in Table 12 below.

Using the full length of the T. viride-derived xylanase gene obtained instep (1)(d) in Example 2 as a template, the DNA sequence of the signalportion was obtained by PCR. The primers used in the PCR are presentedas SEQ ID NO: 69 and SEQ ID NO 70 in Table 12 below.

The DNA sequence of the signal sequence portion and the DNA sequence ofthe xylanase portion were linked together using a PCR method, to obtaina sequence that includes StuI site in a sequence upstream of the startcodon of the signal sequence portion and XhoI site in a sequencedownstream of the stop codon.

The primers used in the PCR are presented as SEQ ID NO: 71 and SEQ ID NO72 in Table 12 below. The amplified 0.7 kbp DNA fragment was insertedinto an expression vector pCR2.1-TOPO using a TOPO TA CLONING KIT(manufactured by Invitrogen Co., Ltd.) according to the protocolattached to the kit, as a result of which a plasmid TOPO-TVX01 wasobtained.

TABLE 12 SEQ ID NO: 67 CAGACGATTGGTCCCGGCACGGGCTTCAACAACGGC TACTSEQ ID NO: 68 CCCCTCGAGTTAGCTGACGGTAATAGAAGCAGAGC SEQ ID NO: 69GGGAGGCCTGCGCATCATGGTTTCCTTCACCTCCC SEQ ID NO: 70GTGCCGGGACCAATCGTCTGGCGCTTTTCAACGTC CACGG SEQ ID NO: 71GGGAGGCCTGCGCATCATGGTTTCCTTCACCTCCC SEQ ID NO: 72CCCCTCGAGTTAGCTGACGGTAATAGAAGCAGAGC

The plasmid TOPO-TVX01 was cleaved with StuI and XhoI, to obtain a genefragment TVX01-N having about 0.7 kbp. Separately, the pCB1-Eg3X-hphless(WO 2011/021616 pamphlet) was cleaved with StuI and XhoI, and a fragmenthaving a length of about 7 kbp was recovered. The recovered fragment waslinked to the gene fragment TVX01-N having a length of about 0.7 kbpusing a DNA ligase (manufactured by Takara Shuzo Co., Ltd.), to producea plasmid TVX01-pCB1. In regard to the reaction conditions for theenzyme and the like, the conditions specified in the instruction manualattached to the kit were adopted. The plasmid TVX01-pCB1 was constructedso as to express TVX01 using its own start codon in the host T. viride.

(b) Production of Transformant of T. viride Using Plasmid TVX01-pCB1

Transformation of T. viride with the plasmid TVX01-pCB1 obtained in step(1)(a) in Example 3 was carried out according to the method disclosed inWO 2011/021616 pamphlet. The transformation was carried out according toa co-transformation method using T. viride strain 2, which is a strainlacking uracil biosynthesis gene (pyr4), as a host and a pyr4 gene ofNeurospora crassa as a selection marker. The T. viride strain 2 can beobtained according to a method disclosed in paragraph number [0102] ofthe specification of Japanese Patent No. 4644603. Specifically, asdescribed in paragraph [0102] of the specification of Japanese PatentNo. 4644603, a spore suspension of the Trichoderma viride MC300-1 strain(FERM BP-6047) at about 10⁹ CFU/ml was irradiated, while gently shaking,with radiation emitted from two UV lamps disposed at a height of 30 cm.The spore suspension after the UV irradiation was applied to a selectionmedium, and cultured for 7 days at 28° C. A strain that grew wasselected, thereby obtaining T. viride strain 2 as a uracil-requiringstrain of Trichoderma viride. The selection medium had a composition ofa minimum medium [0.2% potassium dihydrogen phosphate, 0.4% ammoniumsulfate, 0.03% urea, 0.03% magnesium sulfate heptahydrate, 0.03% calciumchloride, 0.5% glucose, 2.5% agar, and 0.01% trace elements (prepared bydissolving 5 mg iron sulfate heptahydrate, 1.56 mg manganese sulfateheptahydrate, 1.4 mg zinc sulfate heptahydrate, and 2 mg cobalt chloridein 1 L of water)] supplemented with 10 μg/mL uridine and 1 mg/mL5-fluoroorotic acid.

The T. viride strain 2 was suspended in a protoplast-forming enzymesolution (1 mg/mL β-glucuronidase, 0.3 mg/mL chitinase, 0.3 mg/mLZYMOLYASE, and 0.5 mol/L sucrose), to provide protoplasts of themycelia. The obtained suspension was filtered and centrifuged, andthereafter washed with a SUTC buffer solution (0.5 mol/L sucrose, 10mmol/L calcium chloride, and 10 mmol/L Tris-HCl (pH: 7.5)).

The protoplasts were suspended in 100 μL of a SUTC buffer solution, andthen a DNA solution in an amount of 10 μL that contained 10 μg of theplasmid TVX01-pCB1 and a DNA solution in an amount of 10 μL thatcontained the pyr4 gene were added thereto. The resultant mixture wasallowed to stand still in ice for 5 minutes. Next, 400 μL of a PEGsolution (containing PEG4000 at 60%, 10 mmol/L calcium chloride, and 10mmol/L Tris-HCl (pH: 7.5)) was added thereto, and allowed to stand stillin ice for 20 minutes. Then, a SUTC buffer solution in an amount of 10mL was added thereto, and the resultant mixture was centrifuged. Theprotoplasts collected were suspended in 1 mL SUTC buffer solution, and200 μL portions thereof were individually overlaid, together with softagar, on a minimum medium that contained 0.5 mol/L sucrose. Aftercultivation at 28° C. for 5 days, colonies that grew were inoculatedagain into a minimum medium, and the colonies formed therein were usedas transformants.

(c) Cultivation and Identification of Transformant Transformed withTVX01-pCB1

The strains that grew in the minimum medium after the introduction ofthe plasmid TVX01-pCB1 were selected, and cultured according to a methoddisclosed in WO 98/11239. The obtained culture solution was centrifugedto separate culture supernatant from the microbial cells, and theculture supernatant was allowed to pass through a filter (pore size: 0.2μm) for filtration and sterilization, thereby preparing a culturesupernatant solution. The prepared culture supernatant solution wasseparated by electrophoresis using 12% Gel SDS-PAGE mini (manufacturedby TEFKO Co., Ltd.), and a culture supernatant from which a band ofTVX01 was detected well was selected. The selected culture supernatantsolution was named mass production TVX01.

(2) Mass Production of ACX02 Using T. viride

(a) Modification of ACX02 Gene Codon Suitable for Expression in T.viride

In order to enable ACX02 gene to be strongly expressed as an activeprotein in T. viride, a DNA was produced which had changes in bases at24 positions in total in the signal sequence of A. cellulolyticus andACX02 gene. In Table 13, the base positions for the sequence name ACX02refers to the base positions in a wild-type A. cellulolyticus xylanasegene, which is presented as SEQ ID NO: 4. The base positions for thesequence name A. cellulolyticus signal sequence refers to the basepositions in SEQ ID NO: 73.

TABLE 13 Base Before After Sequence Name Position ModificationModification A. Cellulolyticus 12 A C Signal Sequence 42 G T 66 A C 90 GC ACX02 37 A T 38 G C 39 T C 78 T A 81 T C 106 A C 108 G C 138 A C 279 AC 312 A C 405 G C 474 A C 495 A C 552 T C 573 A C 648 G A 663 C G 718 AT 719 G C 720 T C

This modified ACX02 gene was a gene that was designed in considerationof the distribution of the use frequencies of codons in T. viride. Thismodified ACX02 gene was artificially synthesized by Gene Design, Inc. Inthe artificial synthesis, design was performed such that EcoRI and StuIwere included in the sequence upstream of the start codon and such thatXhoI and HindIII were included in the downstream of the stop codon. As aresult, a plasmid pACX02, in which the codon-modified ACX02 gene wasinserted at EcoRI/HindIII of pUC19, was obtained.

(b) Construction of Plasmid ACX02-pCB1

The plasmid pACX02 was cleaved with StuI and XhoI, to obtain a genefragment ACX02-N having a length of about 850 bp. Separately, thepCB1-Eg3X-hphless (WO 2011/021616 pamphlet) was cleaved with StuI andXhoI, and a fragment having a length of about 7 kbp was recovered. Therecovered fragment was ligated to the gene fragment ACX02-N having alength of about 850 bp using a DNA ligase (Takara Shuzo Co., Ltd.), toproduce a plasmid ACX02-pCB1. The reaction conditions for the enzyme andthe like were set to the conditions specified in the instruction manualattached to the kit. The plasmid ACX02-pCB1 had a configuration suchthat ACX02 would be expressed in the host Trichoderma viride using itsown start codon.

(c) Production of Transformant of Trichoderma viride Transformed withPlasmid ACX02-pCB1

Transformation of Trichoderma viride with the plasmid ACX02-pCB1obtained in step (2)(b) in Example 3 was carried out according to themethod disclosed in WO 2011/021616 pamphlet. The transformation wascarried out according to a co-transformation method using T. viridestrain 2, which is strain lacking uracil biosynthesis gene (pyr4), as ahost and a pyr4 gene of Neurospora crassa as a selection marker. TheTrichoderma viride strain 2 was suspended in a protoplast-forming enzymesolution (1 mg/mL of β-glucuronidase, 0.3 mg/mL of chitinase, 0.3 mg/mLof Zymolyase, and 0.5 mol/L of sucrose), to provide protoplasts of themycelia. This suspension was filtered and centrifuged, and thereafterwashed with a SUTC buffer solution (0.5 mol/L sucrose, 10 mmol/L calciumchloride, and 10 mmol/L Tris-HCl (pH: 7.5)).

The protoplasts were suspended in 100 μL of a SUTC buffer solution, andthen a DNA solution in an amount of 10 μL that contained 10 μg of theplasmid ACX02-pCB1 and a DNA solution in an amount of 10 μL thatcontained the pyr4 gene were added thereto. The resultant mixture wasallowed to stand still in ice for 5 minutes. Next, 400 μL of a PEGsolution (containing PEG4000 at 60%, 10 mmol/L calcium chloride, and 10mmol/L Tris-HCl (pH: 7.5)) was added thereto, and allowed to stand stillin ice for 20 minutes. Then, a SUTC buffer solution in an amount of 10mL was added thereto, and the resultant mixture was centrifuged. Theprotoplasts collected were suspended in 1 mL SUTC buffer solution, and200 μL portions thereof were individually overlaid, together with softagar, on a minimum medium that contained 0.5 mol/L sucrose. Aftercultivation at 28° C. for 5 days, colonies that grew were inoculatedagain into a minimum medium, and the colonies formed therein were usedas transformants.

(d) Culturing and Identification of Transformant Transformed withACX02-pCB1

The strains that grew in the minimum medium after the introduction ofthe plasmid ACX02-pCB1 were selected, and cultured according to a methoddisclosed in WO 98/11239.

The obtained culture solution was centrifuged to separate culturesupernatant from the microbial cells, and the culture supernatant wasallowed to pass through a filter (pore size: 0.2 μm) for filtration andsterilization, thereby preparing a culture supernatant solution. Theprepared culture supernatant solution was subjected to SDS-PAGE, and aculture supernatant from which a band of ACX02 was detected well wasselected. The selected culture supernatant solution was named massproduction ACX02.

Example 4 Transition of Stability of TVX01 Along with Changes inTemperature and pH

The following experiment was conducted using the TVX01 mass-produced bythe method employed in Example 3. A 200 mM buffer solution (specifiedbelow) and xylanase were mixed together in a ratio of 1:1, and themixture was treated for a predetermined period of time at varioustemperatures, and then allowed to stand still in an ice bath for 5minutes. Then, residual activity was measured according to the methodemployed in Example 1. The buffer solutions used in this process were asodium citrate buffer solution (pH: 4.5), a Tris-HCl buffer solution(pH: from 8 to 9), and a sodium glycine buffer solution (pH: 10).

The TVX01 retained an activity of 86% even after heat treatment at pH4.5 and 50° C. for 72 hours. In addition, TVX01 retained an activity of68% even after heat treatment at pH 5.5 (the pH of the mutant xylanasestock solution) and at a higher temperature, 70° C., for 5 minutes. Bothconditions are conditions in which wild type would completely lose itsactivity; in contrast, the mutant exhibited improved residual activityunder the acidic, high-temperature conditions.

After heat treatment at 50° C. and a pH of from 8 to 10 for 60 minutes,the residual activity of the wild type was 28% at pH 8, was 8% at pH 9,and complete inactivation was observed at pH 10, whereas the residualactivity of the mutant was 83% at pH 8, was 60% at pH 9, and was 56%even at pH 10. Furthermore, even after heat treatment at 60° C. for 60minutes, in which the wild type completely lose its activity, the mutantxylanase retained an activity of 30% at pH 8, an activity of 17% at pH9, and an activity of 10% at pH 10, which demonstrates that the mutantxylanase exhibits an improved residual activity also under the basic,high-temperature conditions.

As described above, TVX01 according to the invention exhibited asignificant improvement in residual activity under conditions where thewild-type enzyme significantly inactivates, such as pH 4.5 or pH of from8 to 10 with a temperature of from 50° C. to 70° C.

Example 5 Change of Stability of ACX02 Due to Changes in Temperature andpH

An experiment was carried out in the same manner as in Example 4, butusing the ACX02 mass-produced by the method employed in Example 3.

The mass-produced ACX02 retained an activity of 84% even after heattreatment at pH 4.5 and 50° C. for 72 hours. A wild-type xylanasetreated in the same manner retained an activity that was as low as 45%.Thus, it is demonstrated that the mutant xylanase exhibits an improvedresidual activity under the acidic, high-temperature conditions, ascompared to the wild-type xylanase.

The wild type completely lost its activity after heat treatment at 50°C. and pH of from 8 to 10 for 60 minutes, whereas ACX02 retained anactivity of 34% at pH of 8, an activity of 5% at pH 9, and an activityof 2% even at pH 10, which demonstrates that ACX02 retains improvedresidual activity even under the basic, high-temperature conditions.

As described above, ACX02 according to the invention exhibited asignificant improvement in residual activity under conditions where thewild-type enzyme significantly inactivates, such as pH 4.5 or pH of from8 to 10 with a temperature 50° C.

Example 6 Saccharification Reaction (1) of Lignocellulosic Raw Material

Leaf bleached kraft pulp (LBKP) having a dry weight of 2 g was placed inErlenmeyer flasks. Then, a cellulase aqueous solution that contained theACX02 mass-produced by the method of Example 3, a cellulase aqueoussolution that contained the TVX01 mass-produced by the method of Example3, a cellulase aqueous solution that contained a T. viride-derivedwild-type xylanase as an experimental control, and a cellulase aqueoussolution that contained an A. cellulolyticus-derived wild-type xylanaseas an experimental control were individually added into their respectiveErlenmeyer flasks, such that the amount of each cellulase aqueoussolution added was 52 mg in terms of protein weight. Then, 20 mM sodiumcitrate buffer solution (pH 4.5) was added into each Erlenmeyer flask,to prepare a reaction solution in an amount of 20 g. Each of theErlenmeyer flasks was sealed with a silicone plug. After that, thereaction solution was gently stirred at 50° C., and a saccharificationreaction was allowed to proceed. The results are given in Table 14, inwhich WT. represents the wild-type xylanase.

TABLE 14 Name of Residual Activity No. Xylanase [%] (m) WT (T. viride)27 (n) TVX01 90 (o) WT (A. Cellulolyticus) 35 (p) ACX02 90

After 72 hours, the wild-type xylanases derived from A. cellulolyticusand T. viride exhibited residual activities that had decreased to about30% (rows (m) and (o) in Table 14); in contrast, the mass-produced ACX02and the mass-produced TVX01 exhibited residual activities of 90% orhigher (rows (n) and (p) in Table 14).

Example 7 Saccharification Reaction (2) of Lignocellulosic Raw Material

(1) Saccharification Reaction

Leaf bleached kraft pulp (LBKP) having a dry weight of 40 g was placedin separable flasks. Then, a cellulase aqueous solution that containedTVX01 mass-produced by the method of Example 3 and a cellulase aqueoussolution that contained the ACX02 mass-produced by the method of Example3 were individually added, in an amount of 347 mg in terms of proteinweight, into their respective separable flasks. Then, 20 mM sodiumcitrate buffer solution (pH 4.5) was added into each separable flask, toprepare a reaction solution in an amount of 400 g. After that, thereaction solution was gently stirred at 50° C., and a saccharificationreaction was allowed to proceed. The amount of monosaccharide producedafter the reaction was carried out for 72 hours was analyzed by HPLC.

<HPLC Analysis Conditions>

Analyzer: HPLC available from JASCO CorporationColumn: ULTRON PS-80H (300×8 mm; manufactured by Shinwa Chemical Co.,Ltd.)Analysis temperature: 50° C.Mobile phase: Perchloric acid aqueous solution at pH 2.1

(2) Recovery of Enzyme

The saccharification reaction solution (the reaction solution after 72hours of reaction) was centrifuged at 7000×g, and the precipitate wasrecovered. The remaining centrifugal supernatant solution was treatedwith a commercially available UF membrane (product name: Microza UFPencil Module AIP-0013D manufactured by Asahi Kasei ChemicalsCorporation), to obtain a concentrated fraction.

(3) Re-Saccharification Reaction Using Recovered Enzyme

The precipitate obtained by the 7000×g centrifugation of thesaccharification reaction solution and the concentrated fractionobtained by the treatment with the UF membrane were placed in aseparable flask. Next, leaf bleached kraft pulp (LBKP) was added theretoin an amount of 40 g in terms of final solid content, and the resultantmixture was gently stirred at 50° C. and a saccharification reaction wasallowed to proceed. Then, the amount of monosaccharide produced after 72hours of reaction was analyzed by HPLC.

(4) Results of Saccharification Reaction

After 72 hours from the start of the first reaction (the number of timesof enzyme reutilization: zero times), the concentration of glucose andxylose accumulated in the saccharification reaction solution thatcontained TVX01 was 79.1 g/L, in which the concentration of glucoseaccumulated in the saccharification reaction solution was 65.1 g/L. Theconcentration of glucose and xylose accumulated in the saccharificationreaction solution that contained the wild-type T. viride-derivedxylanase was 78.0 g/L, in which the concentration of glucose accumulatedin the saccharification reaction solution was 64.7 g/L. In thesaccharification reaction solution that contained ACX02, theconcentration of glucose and xylose accumulated was 61.3 g/L, in whichthe concentration of glucose accumulated was 49.9 g/L. The enzymescontained in these saccharification reaction solutions were re-utilizedfor saccharification reactions by the method described above.

The concentration of glucose and xylose accumulated in the first cycleof reutilization was 70.7 g/L in the saccharification reaction solutionthat contained TVX01, 53.3 g/L in the saccharification reaction solutionthat contained the wild-type T. viride-derived xylanase, and 53.1 g/L inthe saccharification reaction solution that contained ACX02.

The concentration of glucose accumulated in the first cycle ofreutilization was 58.7 g/L in the saccharification reaction solutionthat contained TVX01, 45.0 g/L in the saccharification reaction solutionthat contained the wild-type T. viride-derived xylanase, and 43.5 g/L inthe saccharification reaction solution that contained ACX02.

The concentration of glucose and xylose accumulated in the second cycleof reutilization was 63.6 g/L in the saccharification reaction solutionthat contained TVX01, 42.3 g/L in the saccharification reaction solutionthat contained the wild-type T. viride-derived xylanase, and 42.6 g/L inthe saccharification reaction solution that contained ACX02.

The concentration of glucose accumulated in the second cycle ofreutilization was 53.0 g/L in the saccharification reaction solutionthat contained TVX01, 35.8 g/L in the saccharification reaction solutionthat contained the wild-type T. viride-derived xylanase, and 34.6 g/L inthe saccharification reaction solution that contained ACX02.

The obtained results are given as relative values assuming that theconcentration of accumulated sugars in the 0th cycle of reutilization is100%. The results are given in Tables 15 and 16, in which WT. representswild-type xylanase.

TABLE 15 Concentration of Glucose and Xylose Name of Accumulated(Relative Values) No. Xylanase 0th Cycle 1st Cycle 2nd Cycle (m) WT (T.viride) 100 68 54 (n) TVX01 100 89 80 (p) ACX02 100 87 70

TABLE 16 Concentration of Glucose Name of Accumulated (Relative Values)No. Xylanase 0th Cycle 1st Cycle 2nd Cycle (m) WT (T. viride) 100 70 55(n) TVX01 100 90 81 (p) ACX02 100 87 69

As demonstrated in Table 15, it was clarified that the mutant xylanasesTVX01 and ACX02 according to the invention are highly suitable for usedin the saccharification reaction in which reutilization of enzymes iscarried out.

As demonstrated in Table 16, it was clarified that not only theefficiency of production of xylose but also the efficiency of productionof glucose is increased by using the mutant xylanase according to theinvention.

Besides, effects similar to the above were obtained also in cases inwhich TVX01 and ACX02 according to the invention were used with needlebleached kraft pulp (NBKP).

Example 8 Method of Bleaching Pulp

(1) Treatment of Pulp with Xylanase

A commercially available milk carton is used as a pulp. The raw materialof milk cartons is timber from thinned softwood, remnant wood generatedby lumbering, or the like, and is a virgin pulp that contains lignin,which is a coloring component.

A well-washed milk carton is cut into about 5-cm square pieces, andimmersed in water for a length of time of from about 2 days to about 5days. Thereafter, a polyethylene film on the surface thereof is removed.

Water in which the paper pieces have been immersed is heated to 50° C.,and the mutant xylanase TVX01 according to the invention and the mutantxylanase ACX02 according to the invention are individually addedthereto. The same treatment is carried out, but using their respectivewild-type xylanases as controls for comparison. Each of the xylanasesused in this example are derived from a filamentous fungus. The amountof xylanase to be added is controlled so as to provide an optimal mixingratio. In addition, the treatment time is also controlled so as toprovide an optimum treatment time. Moreover, a sample that would not betreated with xylanase is also prepared.

After that, a commercially available chlorine-containing bleaching agentis added, and the paper pieces are allowed to stand still for half a dayat a pH of from 7 to 10. The paper pieces are washed well with water,and torn into small pieces, and stirred with an appropriate amount ofwater in a household mixer until the paper pieces become unable to beseen.

The fibers are processed into paper using a commercially availablepapermaking apparatus, and then water is removed therefrom, and thepaper is dried.

(2) Measurement of Whiteness

Whiteness (JIS Z 8715) of the paper produced as described above ismeasured using a UV-visible spectral whiteness meter.

(3) In cases in which the mutant xylanase TVX01 according to theinvention and the mutant xylanase ACX02 according to the invention areadded, pulp can be bleached even under the conditions of pH 7 to pH 10.

Example 9 Detergent

(1) Cleaning of Fluffed Fabric

Old fluffed fabric is used as a material to be washed. The detergent tobe used is prepared by adding the mutant xylanase TVX01 according to theinvention or the mutant xylanase ACX02 according to the invention to acommercially available detergent. Treatment is performed in the samemanner as above, but using their respective wild-type xylanases ascontrols for comparison. Each of the xylanases used in this example isderived from a filamentous fungus. In addition, old fluffed fabric nottreated with xylanase is also prepared. The amount of xylanase to beadded is controlled so as to provide an optimal mixing ratio. Timing ofthe addition thereof is set to be simultaneous with the addition of thedetergent.

800 ml of water is added into a 1 L separable flask, and the detergentand the xylanase are added thereto. Washing is conducted at 50° C. and apH of from 7 to 10 for 1 hour while rotating the separable flask at 60rpm. Thereafter, natural drying is performed.

(2) Measurement of Degree of Removal of Fluff

The state of removal of fluff is observed under a stereomicroscope. Inaddition, the degree of removal of fluff in the fabric after washing ismeasured using a spectrophotometer.

(3) In cases in which the mutant xylanase TVX01 according to theinvention and the mutant xylanase ACX02 according to the invention areadded, fluffing is suppressed under the conditions of a pH of from 7 to10 and a temperature of 50° C.

Example 10 Animal Feed

(1) Production of Animal Feed

The mutant xylanase TVX01 according to the invention or the mutantxylanase ACX02 according to the invention is added to a powdery feed forexperimental animals. Treatment is performed in the same manner butusing their respective wild-type xylanases as controls for comparison.Each of the xylanases used in this example is derived from a filamentousfungus. In addition, powdery feed not treated with xylanase is alsoprepared. The amount of xylanase to be added is controlled so as toprovide an optimal mixing ratio, and the mixture is pelletized.

(2) Measurement of Degree of Cell Wall Decomposition in Shaped AnimalFeed

After the shaped animal feed is allowed to stand still overnight, theanimal feed is sliced with a commercially available razor and subjectedto Gram staining on the prepared slide, and the degree of coloring ofthe cell wall is observed under an optical microscope. In addition, theanimal feed that has been allowed to stand still overnight is vigorouslymixed with 100 mM sodium citrate buffer solution (pH 4.5), andcentrifuged at 5000×g for 15 minutes. Then, the supernatant is removed,and the amount of reducing sugar in the supernatant is measured usingthe DNS method (Bailey et. al, 1992).

Example 11 Bread-Making Modifier

(1) Bread Making

Bread is made using the straight dough method. The formulation ofingredients is given in Table 17 below. For all of the ingredients,commercially available materials for home use are used.

TABLE 17 Name of Amount Added Ingredient (g) Hard flour 320 Milk 100Butter 25 Dry Yeast 4 Salt 5 Sugar 20

The mutant xylanase TVX01 according to the invention or the mutantxylanase ACX02 according to the invention is added as a bread-makingmodifier. Treatment is performed in the same manner but using theirrespective wild-type xylanases as controls for comparison.

The timing of addition thereof is set to be simultaneous with the mixingof ingredients. The amount of xylanase is controlled so as to provide anoptimum mixing ratio. In addition, dough not treated with xylanase isalso prepared.

The dough obtained was allowed to ferment at about 37° C. for a lengthof time of from about 1 hour to about 2 hours until the size thereofincreased to about twice the original size, and then baked in amicrowave.

(2) Observation of Particle Structure of Bread

The baked bread is sliced with a commercially available razor, and theparticle structure is observed under a stereomicroscope.

(3) Measurement of Loaf Volume

The baked bread is allowed to stand still overnight, and then the loafvolume of the baked bread is measured using a rapeseed displacementmethod.

(4) In cases in which the mutant xylanase TVX01 according to theinvention and the mutant xylanase ACX02 according to the invention areadded, the mutant xylanases are capable of stable reaction under theconditions of from 35° C. to 40° C. for from 1 to 2 hours in thefermentation process.

The disclosure of Japanese Patent Application Nos. 2011-257389, filedNov. 25, 2011, and the disclosure of Japanese Patent Application No.2012-099096, filed Apr. 24, 2012, are incorporated herein by referencein their entireties.

All publications, patent applications, and technical standards mentionedin this specification are herein incorporated by reference to the sameextent as if each individual publication, patent application, ortechnical standard was specifically and individually indicated to beincorporated by reference.

1. A nucleic acid encoding a mutant xylanase, the mutant xylanasecomprising the following substitute amino acid residues substitution inthe amino acid sequence of SEQ ID NO: 1: a leucine residue substitutedfor an asparagine residue at position 29; an arginine residuesubstituted for a lysine residue at position 58; an amino acid residue,other than a tyrosine residue, substituted for a tyrosine residue atposition 27; and an amino acid residue, other than an asparagineresidue, substituted for an asparagine residue at position 44, or themutant xylanase comprising 1 to 10 amino acid residues substitution inthe amino acid sequence of SEQ ID NO: 1 in addition of the followingamino acid residues substitutions: a leucine residue substituted for anasparagine residue at position 29, an arginine residue substituted for alysine residue at position 58, a phenylalanine residue substituted for atyrosine residue at position 27, and a serine residue substituted for anasparagine residue at position
 44. 2. The nucleic acid according toclaim 1, wherein the amino acid residue, other than a tyrosine residue,substituted for the tyrosine residue at position 27 in the amino acidsequence of SEQ ID NO: 1 is a phenylalanine residue, and the amino acidresidue, other than an asparagine residue, substituted for an asparagineresidue at position 44 in the amino acid sequence of SEQ ID NO: 1 is aserine residue.
 3. An expression vector comprising the nucleic acidaccording to claim
 1. 4. A transformant comprising the expression vectoraccording to claim
 3. 5. The transformant according to claim 4, whereina host cell of the transformant is a cell derived from Escherichia coli,Bacillus subtilis, yeast, an actinomycete, or a filamentous fungus. 6.The transformant according to claim 5, wherein the filamentous fungusbelongs to the genus Trichoderma, the genus Acremonium, the genusHumicola, or the genus Aspergillus.
 7. The transformant according toclaim 5, wherein the filamentous fungus is Trichoderma viride,Acremonium cellulolyticus, Humicola insolens, or Aspergillus niger.
 8. Amethod of producing a mutant xylanase, the method comprising culturingthe transformant according to claim 4 and recovering the mutant xylanasefrom at least one of the cultured transformant or a culture product ofthe transformant.